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1 From the Department of Internal Medicine and 2 Michigan Diabetes Research and Training Center, University of Michigan Medical School, Ann Arbor.
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Abstract |
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METHODS. Glucose transport in control and VEGF-treated BREC cultures was determined by measurement of [14C]-3-O-methylglucose (3MG) uptake. GLUT1 protein and mRNA was determined by Western and Northern blot analyses, respectively. Protein kinase C (PKC) activity was measured in control and VEGF-treated cultures, and glucose transport was determined with and without prior PKC depletion and PKC inhibition.
RESULTS. Dose-dependent increases in 3MG uptake were seen in the VEGF-treated cultures, with an increase of 69% after a 24-hour exposure to 50 ng/ml VEGF (P < 0.001). Total cellular GLUT1 mRNA or protein, however, was unchanged. Western blot analysis of plasma membrane fractions revealed a 75% increase in plasma membrane GLUT1 in VEGF-treated cultures (P = 0.02), suggesting that the VEGF-stimulated increase in glucose transport was due to a translocation of GLUT1 to the cell membrane. VEGF stimulated a 90% increase in PKC activity in membrane fractions from cultures treated with VEGF, and VEGF-stimulated enhancement of glucose transport was abolished by cellular PKC depletion and by general and PKC ß inhibition.
CONCLUSIONS. The present study demonstrates VEGF-mediated enhancement of retinal endothelial cell glucose transport and suggests that this increase is due to PKC ß–mediated translocation of cytosolic GLUT1 to the plasma membrane surface. Upregulation of retinal endothelial cell glucose transport by various factors associated with the development of retinopathy may be responsible for the metabolic derangements observed in the diabetic inner blood–retinal barrier in vivo.
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Introduction |
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Glucose entry into and through the retinal endothelial cells of the inner blood–retinal barrier (BRB) occurs exclusively through the GLUT1 glucose transporter,9 10 11 12 a member of the sodium-independent glucose transporter family.13 14 GLUT1, which is characteristically expressed in cells that serve barrier functions,9 10 15 16 such as the brain capillary endothelia of the blood–brain barrier (BBB) and the endothelia and retinal pigment epithelium of the inner and outer BRB, is a high-affinity transporter (1–5 mM), and transport through GLUT1 is therefore at near-saturation levels at normal physiological glucose concentrations.17 18 19 20 In the absence of changes in the density of GLUT1 at the endothelial membrane, increasing extracellular glucose concentrations may do little to increase intracellular glucose, a critical molecular event in the development of DR. In contrast, an increase in inner BRB GLUT1 expression may have a profound impact in providing substrate for the molecular processes leading to retinal microvascular disease. In a previous study,21 a dramatic focal increase of GLUT1 abundance was demonstrated on the inner BRB of individuals with long-standing diabetes without clinical evidence of retinopathy. These results suggest that the earliest stages in the development of DR are associated with a localized increase in inner BRB GLUT1 density.
Only a few of the principal factors that modulate inner BRB GLUT1 expression have been identified. Takagi et al.22 have reported recently that in vitro, hypoxia increases GLUT1 protein and mRNA and glucose transport in bovine retinal endothelial cell (BREC) cultures, a process that is regulated by adenosine and the cyclic adenosine monophosphate (cAMP)–PKA pathway. To investigate regulation of retinal endothelial cell glucose transport and GLUT1 expression by growth factors that are known to be associated with the development of DR, we studied the effects of vascular endothelial growth factor (VEGF) on BREC glucose transport and GLUT1 expression. VEGF, a cytokine that has endothelial cell–specific mitogenic and angiogenic properties,23 has been demonstrated to play a major role in the proliferative stages of DR.24 25 Although there is some controversy,26 numerous studies have documented increased retinal VEGF expression in nonproliferative DR as well. In both human diabetic retina27 28 29 and the retina of experimental animal models of DR,30 31 VEGF is increased in the earliest stages of retinopathy, in which it may act to increase microvascular permeability30 and changes in microvascular blood flow.32 In the present study we report a PKC-mediated increase in BREC glucose transport in response to VEGF. Unlike the situation in hypoxia, however, VEGF-mediated increases in glucose transport were not the result of increased total cellular GLUT1 abundance but appeared to follow VEGF-mediated translocation of GLUT1 to the cytoplasmic membrane.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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On the day before each experiment, the medium was changed to DMEM with 0.5% FCS and antibiotics without EGS. The next day, these media were exchanged with either fresh medium alone or medium containing the required concentrations of human recombinant VEGF (R&D Systems, Minneapolis, MN).
Glucose Transport Studies
Uptake of the [14C]-labeled glucose
analogue, 3-O-methylglucose
([14C]-3MG, kindly provided by C. Carter-Su
(University of Michigan), was performed according to the protocol of
Tai et al.,33
with modifications. Briefly, BREC cultures
were grown to near-confluence in 35-cm2 fibronectin-coated
dishes. After treatment with VEGF, the media from control and
VEGF-treated cultures were aspirated and replaced with low-calcium
Krebs–Ringer phosphate buffer containing 1% bovine serum albumin
(KRP-BSA) and incubated at 37°C for 30 minutes to deplete
intracellular glucose concentrations. The cultures were washed twice
with KRP-BSA at room temperature for 5 minutes. The assay was performed
by incubation of each cell culture with 1 ml KRP-BSA containing 0.25
µCi/ml [14C]-3MG (specific activity, 55.2
mCi/mmol) for 7 seconds at room temperature. To assess nonspecific
binding, parallel cultures were preincubated with KRP-BSA containing 40
µM cytochalasin-B (Sigma) for 5 minutes at room temperature, followed
by incubation with the [14C]-3MG solution
containing cytochalasin-B. Maximum uptake was assessed by incubation of
parallel cultures with the radioisotope for 90 seconds. Uptake was
terminated by rapid aspiration of the isotope with repeated washes with
ice-cold phloretin (0.2 mM phloretin in KRP without BSA, Sigma), an
inhibitor of glucose transport. Cells were solubilized in 0.1% SDS and
0.1 M NaOH at 60°C for 30 minutes. Protein was measured by a BCA
assay (Pierce, Rockford, IL). 14C was measured by
liquid scintillation counting (Tri-Carb Scintillation Counter; Packard
Instruments, Downers Grove, IL).
Initial hexose uptake rates were calculated according to the methods of
Carter-Su and Okamoto,34
using the formula devised by
Foley et al.35
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Crude Membrane Preparations
Total cellular membranes were isolated from control and
VEGF-treated BREC cultures according to the methods of Kaiser et
al.36
Briefly, the media of control and VEGF-treated
cultures were aspirated, and the cultures were washed with cold PBS.
Four milliliters of homogenization buffer (0.25 M sucrose, 10 mM
NaHCO3, 5 mM NaN3, and 0.1
mM phenylmethylsulfonyl fluoride) was added to each culture, and the
cells were scraped and transferred to a 10-ml glass tissue grinder. The
cells were homogenized by 60 strokes of a Teflon-coated pestle,
followed by centrifugation at 1200g for 10 minutes at 4°C.
The supernatant was subsequently collected and centrifuged at
9000g for 10 minutes to pellet out cellular organelles and
nuclei. The total cellular membranes in the resultant supernatant were
collected by centrifugation at 100,000g for 60 minutes at
4°C and resuspended in Laemmli loading buffer without
ß-mercaptoethanol (ß-ME).
Plasma Membrane Preparations
Plasma membranes were isolated according to the methods of Jaffe
et al.37
Briefly, the media from control and treated
cultures grown in 150-cm2 flasks were aspirated, and the
cultures were thoroughly washed with ice-cold PBS. Ten milliliters of
ice-cold PBS containing protease inhibitors was added to each flask,
and the cells scraped from two flasks were pooled in a chilled 50-ml
tube. The cells were pelleted, resuspended in PBS with protease
inhibitors and homogenized with 20 strokes in a homogenizer (Dounce;
Kontes Glass, Vineland, NJ). The homogenate was centrifuged at
1800g at 4°C for 10 minutes, and the pellet was
resuspended, homogenized again and centrifuged as before. The two
supernatants were pooled and centrifuged at 30,000g, 4°C,
for 30 minutes. The pellet, which contained nuclei, cellular membranes,
and organelles, was resuspended in 250 mM sucrose in 10 mM Tris,
layered on a discontinuous sucrose gradient, and centrifuged at 55,000
rpm at 4°C for 120 minutes. The interfaces containing the plasma
membranes (i.e., between 20% and 27% and between 32% and 40%) were
carefully aspirated, resuspended in PBS with protease inhibitors, and
collected by another centrifugation at 55,000 rpm at 4°C for 60
minutes. The final pellet was resuspended in Laemmli loading buffer
without ß-ME.
Protein Measurements
Protein concentrations were measured by BCA assay (Pierce) with
BSA as a standard. For Western blot samples, ß-ME was added after
protein measurement to a final concentration of 5%.
Western Blot Studies
For studies involving total cell lysates, electrophoresis was
performed on 25-µg aliquots of each sample on a 10%
SDS-polyacrylamide gel and transferred to polyvinylidene fluoride
(PVDF) membranes. For total cell membranes and plasma membranes,
aliquots of 7.5-µg aliquots were used. An aliquot of 7.5 ng of human
erythrocyte glucose transporter, purified from whole blood as described
previously,38
39
was loaded on each gel for comparison of
molecular mass. Western blot analysis was performed as previously
described40
with a rabbit polyclonal anti-human
erythrocyte glucose transporter antiserum (a kind gift of C. Carter-Su,
University of Michigan) at a concentration of 1:10,000. This antibody
has been characterized previously.33
For the plasma
membrane preparations, Western blot analysis for NaK-ATPase was
performed using a polyclonal rabbit antiserum (UBI, Lake Placid, NY) at
a dilution of 1:4000. After washing, the blots were reacted with an
anti-rabbit secondary antibody coupled to horseradish peroxidase at a
concentration of 1:7500, followed by development with chemiluminescence
reagents (ECL or ECL Plus; Amersham Pharmacia, Piscataway, NJ),
according to the manufacturer’s instructions. Blots were exposed to
radiographic film (X-OMAT; Eastman Kodak, Rochester, NY), and the
autoradiographs were scanned and quantified by computer (NIH Image
software; National Institutes of Health, Bethesda, MD). After
development of the autoradiograms, each Western blot was stained with
Coomassie blue to confirm equal transfer of total proteins to the
membrane.
Northern Blot Studies
Twenty-five-microgram aliquots of total RNA isolated from
confluent 75-cm2 flasks of control and VEGF-treated (50
ng/ml for 24 hours) BREC cultures were isolated (RNeasy; Qiagen,
Valencia, CA), loaded on a 1.1-M formaldehyde 1% agarose gel, and run
overnight at 20 V. RNA was transferred to a membrane (Genescreen Plus;
Dupont–NEN, Boston, MA), and the membrane was dried in a vacuum at
80°C for 2 hours. Northern blot analysis for GLUT1 and mouse actin
were performed as previously described,39
using a 512-kb
PstI fragment of the bovine blood–brain barrier glucose
transporter cDNA41
linearized with HindIII and
with a mouse actin clone, pAM-91 (generously provided by Michael
J. Getz, Mayo Clinic/Foundation, Rochester, MI), linearized
with EcoRI. Both cDNAs were labeled with
[32P]-dCTP using a random primer method, as
described previously.39
PKC Studies
Partially purified PKC was prepared from cytosolic and total
membrane preparations of BREC cultures according to the method of Xia
et al.42
Total PKC activity was determined in the
cytosolic and membrane preparations by measurement of the transfer of
32P from [
-32P]ATP
(100 µCi/mmol) to a PKC-specific peptide
(neurogranin(28–43))43
in the
presence of phosphotidylserine, Ca2+, and
diacylglycerol.42
Involvement of PKC with VEGF-mediated
effects on BREC glucose transport was studied in BREC cultures that had
previously undergone PKC depletion or preincubation with LY379196, a
selective inhibitor of PKC-ß (kindly provided by K. Ways, Eli Lilly,
Indianapolis, IN). Cellular PKC depletion44
was achieved
by overnight incubation of the cultures with 1 µM phorbol
12-myristate 13-acetate (TPA, Sigma) before addition of the growth
factor. The inhibitory profile of LY379196 for various PKC isoforms is
similar to that of LY333531, which has been used in various in vitro
studies of PKC-mediated pathogenic mechanisms in diabetic vascular and
renal complications45
46
47
48
(Table 1)
. LY379196 shows general PKC inhibition at a concentration of
600 nM; at 30 nM, LY379196 demonstrates PKC-ß1 and ß2-selective
inhibition, with median effective doses (ED50) of
0.05 and 0.03 µM, respectively (J. R. Gillig, Eli Lilly). For
PKC inhibition, BREC cultures were preincubated with the inhibitor for
60 minutes at 37°C before the addition of recombinant VEGF.
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Results |
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isoform of NaK-ATPase, which is
localized to the plasma membrane,49
demonstrated a greater
than 20-fold enrichment of the plasma membrane fraction in control
preparations (Fig. 3A
). Quantitative Western blot analysis from control and VEGF-treated
cultures revealed a 75.4% ± 32.0% increase in plasma membrane GLUT1
in the VEGF-treated cultures (P = 0.02, Figs. 3B
3C
).
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, ßI, ßII, and 
have been demonstrated in the rat
retina,50
and and ßII isoforms have been documented
in primary BREC cultures.50
Stimulation of BREC cultures
by VEGF resulted in an increase in total PKC activity in the membrane
fraction of VEGF-treated cultures by 90% (P < 0.01,
Fig. 4
). Total PKC activity in the cytosolic fraction of VEGF-treated cultures
did not change significantly (Fig. 4)
.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The effect of VEGF on retinal endothelial cell glucose transport is not
that of a nonspecific response of cellular metabolism to a mitogen.
Although mitogenic factors such as basic fibroblast growth factor
(bFGF), tumor necrosis factor (TNF)-,52
53
phorbol
esters,44
and transformation54
are known to
cause increased glucose transport and/or GLUT1 abundance in a variety
of cell types, glucose transport in endothelial cells is not responsive
to insulin,53
55
which acts as a major growth factor in
the central nervous system during development.56
Indeed,
GLUT1, which is the predominant glucose transporter in the inner BRB in
vivo,9
10
11
12
is not insulin sensitive.14
Given
evidence that VEGF may act as a survival factor in the retina during
development,57
the ability of VEGF to regulate endothelial
cell glucose transport in conjunction with proliferation may serve to
ensure adequate substrate delivery as well as blood flow during
development. Maintenance of adequate nutrient transport to the retina
during development and after maturation is of critical importance,
because neuroretinal metabolism is completely dependent on glucose.
VEGF increases retinal endothelial cell glucose transport, not through an increase in total cellular GLUT1 transcript and protein, but by an apparent translocation of preexisting cytoplasmic transporters to the plasma membrane (Fig. 3) . In this sense, the actions of VEGF on GLUT1 are similar to those of insulin on GLUT4 in insulin-sensitive tissues.58 The VEGF-stimulated increase in glucose transport in the absence of an increase in total cellular abundance of GLUT1 in retinal endothelial cells is in apparent contrast to its effects on primary cultures of bovine aortic endothelial cells (BAECs), in which exposure to comparable concentrations of VEGF results in an approximate threefold increase in 2-deoxyglucose uptake and a fivefold increase in GLUT1 transcript.53 In the present studies, exposure of BREC to VEGF at comparable concentrations for up to 24 hours did not result in a statistically significant difference in GLUT1 mRNA compared with control cultures (Fig. 2C) . Differential effects of VEGF on aortic and retinal endothelial cells were not directly compared in these studies, which concentrated on the effects of this cytokine on glucose transport in a microvascular endothelial cell type associated with diabetic complications. Nonetheless, one may speculate that the discrepancy in the results of the present study with those reported by Pekala et al.53 may be due to inherent differences in endothelia isolated from microvascular versus macrovascular sources. In this regard, Thieme et al. 59 have demonstrated that although BRECs and BAECs possess the same types of high-affinity receptors for VEGF, BRECs possess a threefold higher density of these receptors than do BAECs. The differences in VEGF receptor abundance, or perhaps the relative levels of expression of the different receptors for VEGF, in retinal and aortic endothelia may account for the different VEGF-mediated responses in glucose transport and GLUT1 expression in these two cell types.
Activation of PKC by hyperglycemia, presumably through de novo synthesis by diacyl glycerol, has been proposed as one of the principal biochemical pathways responsible for the development of diabetic microvascular complications.3 The actions of VEGF in binding to its receptors on endothelial cell membranes are in part mediated by activation of PKC.46 These actions include changes in retinal blood flow,32 microvascular permeability,47 and endothelial cell mitogenesis.46 The present study demonstrates that VEGF-mediated increases in retinal endothelial cell glucose transport occur through activation of PKC. This conclusion is supported by increased localization of PKC to the plasma membrane in VEGF-stimulated BREC cultures (Fig. 4) and abrogation of VEGF-stimulated increases in glucose transport by depletion of PKC intracellular stores (Fig. 5A) and by generalized inhibition of PKC (Fig. 5B) . The observation of the present study that VEGF increases PKC activity in BREC cultures is in close agreement with that of Xia et al.,46 who have documented similar effects in bovine aortic endothelial cells. Furthermore, the demonstration of the ability of the ß isoform–selective inhibitor LY379196 to abolish VEGF-stimulated increases in BREC glucose transport (Fig. 5B) suggests that VEGF’s actions in modulating retinal endothelial glucose transport are mediated by PKC-ß, the PKC isoform that is thought to be responsible for characteristic changes in retinal blood flow45 and microvascular permeability47 observed in experimental models of diabetes.
The principal factors modulating retinal endothelial cell GLUT1 expression have yet to be fully elucidated. In a recent publication, Takagi et al.22 have demonstrated that hypoxia causes an eightfold increase in GLUT1 mRNA and two- and threefold increases in 2-deoxyglucose transport and immunoreactive GLUT1, respectively, in BREC cultures after a 12-hour exposure to hypoxic conditions. With regard to the direct effect of glucose on retinal endothelial glucose transport and GLUT1 expression, Mandarino et al.60 have reported no change in 3MG transport in BREC cultures exposed to elevated glucose concentrations for 5 days. In Mandarino et al., however, changes in the abundance of BREC GLUT1 mRNA and protein were not reported. Nonetheless, focal upregulated immunoreactive GLUT1 expression has been documented in the human diabetic inner BRB.21
Because hyperglycemia per se does not appear to cause an increase in glucose transport nor in GLUT1 expression, it is unlikely that hyperglycemia-mediated changes in glucose transport and/or GLUT1 expression represent the initiating event in the molecular processes underlying the development of DR. Although speculative at this point, it is possible that in the setting of long-standing diabetes, interactions of growth factors or advanced glycation end products with their respective endothelial cell receptors61 62 or the interaction of these receptors with cell surface integrins63 64 may initiate processes that upregulate glucose transport and GLUT1 expression on the endothelial cell surface. This increase in glucose flux into the endothelia of the inner BRB may have toxic effects on the endothelial cells by exposure of the intracellular environment to elevated glucose concentrations. We propose that hypoxia, elevated VEGF production, and other as yet unidentified factors associated with the development of diabetic retinopathy contribute to causing an upregulation of glucose transport in the endothelial cells of the diabetic inner BRB and that this enhancement exacerbates the deleterious effects of hyperglycemia on the retinal microvasculature.16
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Acknowledgements |
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Footnotes |
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Submitted for publication May 27, 1999; revised December 29, 1999; accepted January 26, 2000.
Commercial relationships policy: N.
Corresponding author: Arno K. Kumagai, Department of Internal Medicine, 5570 MSRB-2, University of Michigan Medical School, Ann Arbor, MI 48109-0678. akumagai{at}umich.edu
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