GLUT-1 Expression in Bovine Retinal Capillary Endothelial Cells and Pericytes Exposed to Advanced Glycation End Products

Abstract

Purpose.: Glucose uptake and glucose transporter GLUT-1 expressions are characteristic of retinal capillary endothelial cells and pericytes in response to high glucose. In this study, the effects of advanced glycation end product (AGE-BSA) exposure on these parameters were tested.

Methods.: Primary cultures of bovine retinal capillary endothelial cells (BRECs) and bovine retinal capillary pericytes (BRPs) were exposed to AGE-BSA (100 μg/mL) for 6 days. Glucose uptake was measured using U ¹⁴C-glucose and the GLUT-1 mRNA expression by RT-PCR. GLUT-1 protein was detected by immunofluorescence and subjected to FACS analysis.

Results.: The authors observed that there was no significant decrease in the GLUT-1 protein expression, and this was confirmed by glucose uptake by ¹⁴C-labeled glucose in both BRECs and BRPs. Even though there was a slight decrease in the mRNA expression of GLUT-1 in AGE-BSA–treated cells compared with both untreated control and BSA treated, the decrease was not significant.

Conclusions.: This is the first report to show that there is no difference in glucose uptake in BRECs and BRPs on exposure to AGE-BSA.

The retinal microvasculature is composed of endothelial cells (ECs) and pericytes that line the vessel and rest on a single basement membrane, forming the inner blood retinal barrier. The cellular and biochemical abnormalities responsible for the pericyte loss and endothelial dysfunction in diabetic retinopathy (DR) are still under investigation. Studies emphasize the role of tight control of glucose to reduce the complication of diabetes. ^1,2  

Advanced glycation end products (AGE), a heterogeneous group of compounds reportedly elevated in diabetes, have been implicated in microvascular complications of DR by promoting EC barrier permeability, proliferation, migration, and elaboration of growth factors. ¹ In addition, AGE promotes apoptotic signaling in pericytes, the loss of which is associated with DR. ^2,3 The glucose transport in retinal ECs occurs by the carrier-mediated facilitated transport, independent of insulin. GLUT-3 mRNA and protein have been identified in human retinal ECs, ^4,5 but the GLUT-3 protein and mRNA expression level were shown to be unaffected in the high-glucose environment. ^6,7 However, GLUT-1 is shown to be downregulated in high-glucose conditions. ^7,8 Kumagai et al. ⁹ have shown the absence of GLUT-1 in the ECs of proliferative neovascular vessels in PDR compared to immunopositivity of the retinal ECs. Proliferative diabetic retinopathy (PDR) is a complication of diabetes mellitus characterized by preretinal neovascularization and the development of epiretinal fibrovascular traction, leading to retinal detachment. Although a high-glucose–mediated downregulation of GLUT-1 has been shown in pericytes, no such change was reported in ECs. ¹⁰ However, brain ECs have shown a downregulation of GLUT-1 expression unlike that of retinal ECs, indicating that retinal ECs can be metabolically different from brain ECs. ⁶  

The effect of AGE on the biochemical process of glucose uptake and the GLUT-1 translocation in bovine retinal endothelial cells (BRECs) and bovine retinal pericyte (BRP) cells have not been reported so far. Therefore, the objective of this study was to determine the glucose uptake and GLUT-1 expression in BRECs and BRPs in response to AGE. 

Materials and Methods

Antibodies and other materials used were as follows: fetal bovine serum (Gibco, Grand Island, NY), factor VIII antibody (Dako, Glostrup, Denmark), actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), NG2, VE-cadherin and carboxyl methyl lysine antibody (Chemicon, Temecula, CA), GLUT-1 primary antibody raised in goat, FITC-conjugated anti–goat secondary antibody (Santa Cruz Biotechnology).¹⁴C- labeled glucose was obtained from Bhabha Atomic Research Centre (BARC; Mumbai, India). Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and GLUT-1 were designed using Primer 3 software and were synthesized from Bangalore Genei (Karnataka, India), and complete medium for culturing retinal ECs was used (Endopan media; PAN Biotech, Aidenbach, Germany). DMEM/F12 media was obtained from Gibco. 

Cell Culture

Primary cultures of BRECs and BRPs were isolated from the bovine retina by homogenization of the bovine retinal capillaries under sterile conditions, followed by filtration through a 41-μm nylon filter for BRECs and through a 60 μm filter for BRP. To further purify the isolated BRECs, CD31 antibody–coated beads (Dynabeads; Invitrogen, Carlsbad, CA) were used. Cells were cultured in 5% CO₂ at 37°C, and the media were changed every 3 days. Primary culture of BRECs was grown on 0.1% gelatin coated Petri dish using commercial media for growing ECs (Endopan media; Genex) containing 20% FBS. The BRPs were grown in DMEM/F12 (containing 17.5 mM glucose) + 10% FBS. BREC and BRP populations were confirmed by monitoring the expression of cell surface markers specific to each cell type. Thus, the purity of BRECs was confirmed by immunoreactivity with factor VIII and VE-cadherin, whereas the purity of BRPs was confirmed by immunoreactivity with NG2 and Actin. Cells from passages 2 to 4 were used for the experiments. 

Preparation of Advanced Glycation End Products

AGE-BSA was prepared by slightly modifying the method of Yamagishi et al. ¹¹ and incubating 0.5 M glucose with 50 mg/mL BSA in PBS for 6 weeks at 37°C. For the control, BSA without glucose was incubated under similar conditions. The preparation was eluted through a column (PD-10; Amersham, Buckinghamshire, UK) with PBS to remove the excess salts and unreacted glucose. The glycated adduct formed was then confirmed by 7.5% SDS-PAGE to compare the BSA alone (control) and the glucose-treated BSA for AGE-BSA formation by Western blot for carboxy methyl lysine (CML). ¹²  

MTT Assay to Test for Cytotoxic Effects of AGE-BSA on BRECs and BRP Cells

BRECs and BRPs were grown independently in a 96-well plate (1000 cells/well) and were exposed to various concentrations of AGE-BSA (50, 100, and 200 μg/mL) for 6 days in DMEM/F12 + 1% FBS with a change of media every 48 hours. Formazan, formed after treatment with MTT, was dissolved in dimethyl sulfoxide (DMSO) and read at 570 nm to assess cell viability. 

Fluorescence Labeling of GLUT-1 in BRECs and BRP Cells

BRECs and BRPs were grown in coverslips in a 24-well plate and exposed to AGE-BSA (100 μg/mL) for 6 days with a change of media every 48 hours. BSA-treated cells and cells unexposed to AGE-BSA were used as controls. After 6 days, the cells were serum starved and glucose starved, as discussed for the uptake experiment, and were exposed to 5 mM glucose for 5 seconds (BRECs) and 10 seconds (BRPs). The cells were fixed in 100% methanol and stained for GLUT-1 protein (Santa Cruz Biotechnology). The fluorescent cells were observed under a fluorescence microscope (AxioVision; Carl Zeiss, Oberkochen, Germany). 

Fluorescence-Activated Cell Sorting for GLUT-1 in BRECs and BRPs

For each experiment 30,000 cells were plated in a 25-cm² flask and allowed to reach 50% confluence. The cells were then exposed to 100 μg/mL AGE-BSA for 6 days, with a change of media every 48 hours. Controls were the untreated cells unexposed to AGE-BSA and cells treated with 100 μg/mL BSA alone. At the end of the sixth day, the same cell treatment procedure was followed, and 5 mM glucose was added and incubated for 5 seconds for BRECs and 10 seconds for BRPs, respectively. The uptake was stopped by the addition of ice-cold PBS followed, by profuse washing (three times) of cells in ice-cold PBS. Then the cells were detached using 0.1% trypsin EDTA and were centrifuged. BSA (1%) and sodium azide (1%) were added to the pellet and incubated for half an hour, after which 100 μL of 1:50 diluted polyclonal anti–bovine GLUT-1 raised in goat (Santa Cruz Biotechnology) was added. The cells were again washed in PBS and incubated with 1:100 dilution of anti–goat tagged with FITC (Santa Cruz Biotechnology) in 3% BSA + 1% sodium azide. The cells were again washed in PBS three times and were fixed in 0.1% paraformaldehyde for flow cytometric analysis. Control cells, which were not treated with primary antibody, were used to set the voltage, and those cells that were treated with secondary antibody alone were used to set the background. Flow cytometric analysis was performed in a four-color flow cytometer (FACSCalibur, model E97600177; BD Biosciences, Franklin Lakes, NJ). Data acquisition and analysis were performed (CellQuest Pro software; BD Biosciences), and data were expressed as mean fluorescence intensity (MFI). 

U ¹⁴C–labeled Glucose Uptake in BRECs and BRPs

BRECs and BRPs were plated in six-well plates (15,000 cells/well) and were allowed to grow until 50% confluence in DMEM-F12 with 10% FBS. The cells were then exposed to 100 μg/mL AGE-BSA for 6 days, with fresh media added every 48 hours. Cells with BSA and without any treatment were used as controls. At the end of the sixth day, the cells were serum depleted (with 1% FBS + PBS) for 1 hour; this was followed by glucose depletion for another hour (with 1% BSA + PBS). Then 0.25 μCi of U ¹⁴C glucose was added, and the cells were incubated (5 seconds for BRECs, 10 seconds for BRPs). The kinetics of glucose uptake was 5 to 10 seconds in BRECs and 10 to 15 seconds in BRPs, as reported earlier. ^10,13 Glucose uptake was stopped by the addition of ice-cold PBS, followed by washing (three times) in ice-cold PBS to remove residual radioactivity. The cells were solubilized using 0.1% SDS with 0.1% NaOH; 200 μL solubilized cell lysate was added to 2 mL scintillation fluid, and the disintegrations per minute were counted in the liquid scintillation system (Beckman-6500; Beckman-Coulter, Fullerton, CA). 

Expression of GLUT-1 in BRECs and BRPs by RT-PCR

Total RNA was extracted (Genelute mammalian total RNA mini prep kit; Sigma, St. Louis, MO) according to the manufacturer's instructions. For RT-PCR, 1 μg total RNA was treated with DNase I (Invitrogen), and reverse transcription was carried out using random hexamer (Thermoscript; Invitrogen) using the manufacturer's protocol. PCR was carried out using the following primers: for bovine GAPDH, forward primer 5′-TGTTCCAGTATGATTCCACCC-3′ and reverse primer 5′-GTCTTCTGGGTGGCAGTGAT-3′ corresponding to 424 bp; for GLUT-1, forward primer 5′-TCCTGCTGC CCTTCTGCCCC-3′ and reverse primer 5′-AGGATGGGCTGGCGGTAGGC-3′ corresponding to174 bp. The bands obtained were quantified using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html) after normalization to GAPDH. 

Statistical Analysis

Determinations were performed in duplicate, and experiments were repeated at least three times. Results were expressed as mean ± SEM unless otherwise indicated. Statistical analysis was performed using Wilcoxon test (SPSS version 14; SPSS, Inc., Chicago, IL). 

Results

BRECs and BRP cultures were established, and cells from passages 2 to 4 were used in all experiments. The formation of glycated BSA was seen by increased apparent molecular weight of the band, as seen in 7.5% SDS PAGE and as confirmed by Western blot analysis for the glycation moiety for CML with specific antibody. 

MTT Assay

The effect of AGE-BSA in terms of varying concentrations (50, 100, and 200 μg/mL) on the sixth day of exposure to BRECs and BRPs, showed that the cells were viable until 6 days of AGE-BSA exposure (Fig. 1); 100 μg AGE-BSA was used for the rest of the experiments. 

Figure 1.

 

MTT of BRECs and BRPs with varying concentrations of AGE-BSA (50, 100, and 200 μg) on the sixth day. The experiment was performed in duplicate at least three times.

GLUT-1 Protein Expression by Immunoreactivity

There was a decrease in the expression of GLUT-1 by immunofluorescence in the AGE-BSA–treated cells compared with BSA-treated cells and the untreated controls in both the BRECs and the BRPs on the sixth day of AGE-BSA exposure (Fig. 2). 

Figure 2.

 

GLUT-1 expression in BRECs and BRPs by immunofluorescence. (A) BRECs: untreated control cells; BSA (100 μg/mL)–treated cells; AGE-BSA (100 μg/mL)–treated cells. (B) BRPs: untreated control cells; BSA (100 μg/mL)–treated cells; AGE-BSA (100 μg/mL)–treated cells. Green immunofluorescence indicates GLUT-1 expression.

GLUT-1 Translocation

Because we observed a decrease in GLUT-1 protein expression by immunofluorescence, we analyzed the GLUT 1 protein function by ¹⁴C-labeled glucose uptake in both BRECs and BRPs exposed to AGE-BSA. Although their seemed to be a decrease in the glucose uptake in BRECs, statistical analysis revealed there was no significance. No significant change was observed in glucose uptake in the BRPs in AGE-BSA treated cells compared to BSA-treated and untreated control (Fig. 3). To see the translocation of the GLUT-1 protein in response to AGE-BSA treatment in BRECs and BRPs, FACS analysis was conducted. Similar to the observation in glucose uptake, we found that there is no statistically significant decrease in GLUT-1 expression in BRECs and no changes in BRPs (Figs. 4A, 4B). 

Figure 3.

 

¹⁴C-labeled glucose uptake in BRECs and BRPs. ¹⁴C-labeled glucose uptake in BRECs and BRPs after 6 days of AGE-BSA (100 μg/mL) treatment. Experiments were repeated in duplicate at least three times. Results are expressed normalizing to percentage of control.

Figure 4.

 

GLUT-1 expression by FACS in the BRECs and BRPs treated with AGE-BSA. (A) BRECs. (B) BRPs. Data are expressed as mean ± SEM. Experiments were performed in duplicate and repeated at least three times.

mRNA Expression of GLUT-1 in BRECs and BRPs

The mRNA expression of GLUT-1 by RT-PCR was also done and normalized to GAPDH, but there was no marked change in both the cells when compared to cells treated with BSA (Fig. 5). 

Figure 5.

 

mRNA expression of GLUT-1 by RT-PCR (A, B) GLUT-1 mRNA expression in BRECs. (A) Lane 1, negative control; lane 2, control GAPDH; lane 3, BSA GAPDH; lane 4, AGE-BSA GAPDH; lane 5, control GLUT-1; lane 6, BSA GLUT-1; lane 7, AGE-BSA GLUT-1; lane 8, molecular weights: GLUT-1, 174 bp; GAPDH, 424 bp. (B) Histogram based on band intensity, showing the expression of GLUT-1 in BRECs normalized to GAPDH. (C, D) GLUT-1 mRNA expression in BRPs. (C) Lane 1, negative control; lane 2, control GLUT-1; lane 3, BSA GLUT-1; lane 4, AGE-BSA GLUT-1; lane 5, control GAPDH; lane 6, BSA GAPDH; lane 7, AGE-BSA GAPDH; lane 8, molecular weights: GLUT-1, 174 bp; GAPDH, 424 bp. (D) Histogram based on band intensity, showing the expression of GLUT-1 in BRPs normalized to GAPDH.

Discussion

High glucose levels lead to formation of various types of advanced glycation end products, both at the intracellular and the extracellular levels, and have been reported to be associated with the microvascular complications of diabetes through various mechanisms. ¹⁴ Current studies are focused on cellular changes in the retinal capillaries to understand the pathophysiology of diabetic retinopathy. Among the five different isoforms of glucose transporters, GLUT-1 and GLUT-3 are the most widely distributed and regulate basal glucose uptake in the ECs of barrier tissues. ^15,16 In this study, we found that there was no change in glucose uptake in both cells when they were exposed to AGE-BSA. Takagi et al. ¹⁷ have demonstrated that in conditions such as hypoxia, there is a significant increase in glucose uptake and GLUT-1 mRNA expression in BREC cultures. This may be due to VEGF because BRECs showed an increase in translocation of GLUT-1 to the membrane, when treated with VEGF, resulting in increased glucose uptake. ¹³ Moreover, GLUT-1 expression is shown to differ between retina and cerebral cortex, suggesting that glucose transport is regulated differently in these embryologically similar tissues. ¹⁸ With regard to the type of barrier EC, Rajah et al. ⁶ have shown that there is downregulation of GLUT-1 expression in brain ECs but that there is no such downregulation, even after exposure to high glucose for 5 days, in retinal ECs. In this study we report that there is no significant change in glucose uptake and GLUT-1 expression in response to AGE-BSA, as observed for high glucose and hypoxia conditions in bovine retinal ECs. 

Altered growth rate, DNA fragmentation, priming of the apoptotic signal in response to glucose fluctuation, and high glucose have been reported in pericytes. ¹⁹ Downregulation of the GLUT-1 protein is also reported in pericytes under high-glucose conditions. ¹⁰ This study, for the first time, reports there is no decrease in glucose transport and GLUT-1 expression in response to AGE-BSA in pericytes. Additional studies are needed to look at the specific transcription factors involved in the regulation of GLUT-1 expression. 

Footnotes

 Supported by the Indian Council of Medical Research, Government of India Grant 52/18/2002-BMS.

Footnotes

 Disclosure: S. Barathi, None; N. Angayarkanni, None; V.N. Sumantran, None