Glucose-induced activation of glucose uptake in cells from the inner and outer blood-retinal barrier

Julia V. Busik, L. Karl Olson, Maria B. Grant, Douglas N. Henry

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

PURPOSE. The purpose of this study was to elucidate in vitro the effect of elevated glucose on glucose uptake in the cells comprising the inner and outer blood-retinal barriers: human retinal pigment epithelial (hRPE) and human retinal vascular endothelial (hRVE) cells. METHODS. Primary cultures of hRPE and hRVE cells grown in 5.5 or 22 mM glucose or in 22 mM mannitol were used to measure the rates of glucose uptake with [3H]-3-0-methyl glucose as a tracer. GLUT1 expression was measured by Northern and western blot analyses. Cellular fractionation was performed by differential centrifugation. GLUT1 overexpression was accomplished by adenoviral transduction. RESULTS. Increasing media glucose from 5.5 to 22 mM for 30 minutes caused a 1.9-fold increase in the Vmax of glucose uptake in hRPE cells and a 2.5-fold increase in hRVE cells. These increases were nonosmotic and glucose specific, in that the exposure to 22 mM mannitol did not affect the Vmmax of glucose uptake. mRNA, total protein expression, and translocation of GLUT1, the glucose transporter predominantly expressed in hRPE and hRVE cells, were not affected by 22 mM glucose for up to 48 hours. High-glucose effects on Vmax were abolished with 10 μg/mL of the microtubule assembly inhibitor nocodazole. hRPE cells transduced to overexpress GLUT1 showed a 1.5-fold increase in the Vmax for glucose uptake versus control-transduced cells. However, the magnitude of glucose-induced increase in glucose uptake was the same in GLUT1- and control-transduced cells. CONCLUSIONS. High glucose induced 1.9- and 2.5-fold increases in the Vmax of glucose uptake in hRPE and hRVE cells, respectively. These increases were not due to an increase in GLUT1 expression. The increases were dependent on microtubule integrity, but not on GLUT1 translocation. The mechanism of the increases is unknown. GLUT1 regulating protein(s) and/or novel glucose transporter(s) may be involved in the regulation of glucose uptake by glucose in the cells that comprise the blood-retinal barrier.

Original languageEnglish (US)
Pages (from-to)2356-2363
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number7
StatePublished - 2002
Externally publishedYes

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Blood-Retinal Barrier
Glucose
Retinal Pigments
Retinal Vessels
Endothelial Cells
Facilitative Glucose Transport Proteins
Mannitol
Microtubules
Glucose Transporter Type 1
Epithelial Cells
Nocodazole

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Glucose-induced activation of glucose uptake in cells from the inner and outer blood-retinal barrier. / Busik, Julia V.; Olson, L. Karl; Grant, Maria B.; Henry, Douglas N.

In: Investigative Ophthalmology and Visual Science, Vol. 43, No. 7, 2002, p. 2356-2363.

Research output: Contribution to journalArticle

Busik, Julia V. ; Olson, L. Karl ; Grant, Maria B. ; Henry, Douglas N. / Glucose-induced activation of glucose uptake in cells from the inner and outer blood-retinal barrier. In: Investigative Ophthalmology and Visual Science. 2002 ; Vol. 43, No. 7. pp. 2356-2363.
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AU - Busik, Julia V.

AU - Olson, L. Karl

AU - Grant, Maria B.

AU - Henry, Douglas N.

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N2 - PURPOSE. The purpose of this study was to elucidate in vitro the effect of elevated glucose on glucose uptake in the cells comprising the inner and outer blood-retinal barriers: human retinal pigment epithelial (hRPE) and human retinal vascular endothelial (hRVE) cells. METHODS. Primary cultures of hRPE and hRVE cells grown in 5.5 or 22 mM glucose or in 22 mM mannitol were used to measure the rates of glucose uptake with [3H]-3-0-methyl glucose as a tracer. GLUT1 expression was measured by Northern and western blot analyses. Cellular fractionation was performed by differential centrifugation. GLUT1 overexpression was accomplished by adenoviral transduction. RESULTS. Increasing media glucose from 5.5 to 22 mM for 30 minutes caused a 1.9-fold increase in the Vmax of glucose uptake in hRPE cells and a 2.5-fold increase in hRVE cells. These increases were nonosmotic and glucose specific, in that the exposure to 22 mM mannitol did not affect the Vmmax of glucose uptake. mRNA, total protein expression, and translocation of GLUT1, the glucose transporter predominantly expressed in hRPE and hRVE cells, were not affected by 22 mM glucose for up to 48 hours. High-glucose effects on Vmax were abolished with 10 μg/mL of the microtubule assembly inhibitor nocodazole. hRPE cells transduced to overexpress GLUT1 showed a 1.5-fold increase in the Vmax for glucose uptake versus control-transduced cells. However, the magnitude of glucose-induced increase in glucose uptake was the same in GLUT1- and control-transduced cells. CONCLUSIONS. High glucose induced 1.9- and 2.5-fold increases in the Vmax of glucose uptake in hRPE and hRVE cells, respectively. These increases were not due to an increase in GLUT1 expression. The increases were dependent on microtubule integrity, but not on GLUT1 translocation. The mechanism of the increases is unknown. GLUT1 regulating protein(s) and/or novel glucose transporter(s) may be involved in the regulation of glucose uptake by glucose in the cells that comprise the blood-retinal barrier.

AB - PURPOSE. The purpose of this study was to elucidate in vitro the effect of elevated glucose on glucose uptake in the cells comprising the inner and outer blood-retinal barriers: human retinal pigment epithelial (hRPE) and human retinal vascular endothelial (hRVE) cells. METHODS. Primary cultures of hRPE and hRVE cells grown in 5.5 or 22 mM glucose or in 22 mM mannitol were used to measure the rates of glucose uptake with [3H]-3-0-methyl glucose as a tracer. GLUT1 expression was measured by Northern and western blot analyses. Cellular fractionation was performed by differential centrifugation. GLUT1 overexpression was accomplished by adenoviral transduction. RESULTS. Increasing media glucose from 5.5 to 22 mM for 30 minutes caused a 1.9-fold increase in the Vmax of glucose uptake in hRPE cells and a 2.5-fold increase in hRVE cells. These increases were nonosmotic and glucose specific, in that the exposure to 22 mM mannitol did not affect the Vmmax of glucose uptake. mRNA, total protein expression, and translocation of GLUT1, the glucose transporter predominantly expressed in hRPE and hRVE cells, were not affected by 22 mM glucose for up to 48 hours. High-glucose effects on Vmax were abolished with 10 μg/mL of the microtubule assembly inhibitor nocodazole. hRPE cells transduced to overexpress GLUT1 showed a 1.5-fold increase in the Vmax for glucose uptake versus control-transduced cells. However, the magnitude of glucose-induced increase in glucose uptake was the same in GLUT1- and control-transduced cells. CONCLUSIONS. High glucose induced 1.9- and 2.5-fold increases in the Vmax of glucose uptake in hRPE and hRVE cells, respectively. These increases were not due to an increase in GLUT1 expression. The increases were dependent on microtubule integrity, but not on GLUT1 translocation. The mechanism of the increases is unknown. GLUT1 regulating protein(s) and/or novel glucose transporter(s) may be involved in the regulation of glucose uptake by glucose in the cells that comprise the blood-retinal barrier.

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