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Glut1 and Glut3 expression, but not capillary density, is increased by cobalt chloride in rat cerebrum and retina
Molecular Brain Research 64 Ž1999. 24–33
Research report
Glut1 and Glut3 expression, but not capillary density, is increased by cobalt chloride in rat cerebrum and retina Gamal A. Badr, Jin-Zhong Zhang, Jie Tang, Timothy S. Kern, Faramarz Ismail-Beigi
)
Departments of Medicine and Physiology and Biophysics, and Diabetes Research Center, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106-4951, USA Accepted 27 October 1998
Abstract Treatment of rats with cobalt chloride wCoŽII.x, an agent that stimulates the expression of a set of hypoxia-responsive genes, for 10–12 days resulted in 1.45- and 1.40-fold increases in the content of Glut1 mRNA and Glut1 in cerebral gray matter, respectively Ž P - 0.05 for both changes.. The increase in Glut1 content was associated with a significant increase in the content of Glut1 staining in microvessels isolated from cerebral gray matter, and in the intensity of Glut1 in microvessels of the frontal lobe and hippocampus assessed by immunohistochemistry. The abundance of Glut3 in cerebrum of CoŽII.-treated rats also increased by 1.3-fold Ž P - 0.05., but the increase was not associated with a change in the content of Glut3 mRNA. In retina, treatment with CoŽII. resulted in 2.48- and 1.23-fold increases in the content of Glut1 mRNA and Glut1 protein, respectively Ž P - 0.05 for both changes.; similar increases in Glut1 protein expression were observed in isolated retinal microvasculature. The content of Glut3 in retina also increased 1.5-fold in CoŽII.-treated rats Ž P - 0.05.. In addition, treatment with CoŽII. resulted in a significant 2.2-fold increase in the expression of VEGF in the cerebrum. However, despite the CoŽII.-induced increase in Glut1 expression in cerebral and retinal microvasculature and VEGF in cerebrum, there was no increase in the capillary density in either tissue. It is concluded that a 10–12 day exposure to CoŽII., presumably acting through the hypoxia-signaling pathway, results in enhanced expression of both major glucose transporters in cerebral cortex and retina, without increasing the capillary density of either tissue. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Blood–brain barrier; Blood–retinal barrier; Glut1 mRNA; Glut3 mRNA; Competitive PCR; Brain microvessel; Retinal microvasculature; Capillary density; Hypoxia; VEGF
1. Introduction The metabolic activity of the central nervous system and the retina is critically dependent on metabolism of glucose as the predominant energy substrate w5,13x. Glucose transport across plasma membranes is mediated by a family of six sodium-independent glucose transporter glycoprotein molecules ŽGluts., which manifest distinct kinetic properties and tissue-specific patterns of expression w24x. In brain, Glut1 is highly expressed in endothelial cells of the blood–brain barrier ŽBBB. and to the choroid plexus epithelial cells w12,17,34x. Glut3, the other major Glut isoform present in the brain, is expressed at very high levels in neuronal cells, but not in brain microvessels w9,14,24x. In retina, Glut1 is highly expressed in pigment ) Corresponding author. Clinical and Molecular Endocrinology, Case Western Reserve University, Cleveland, OH 44106-4951, USA. Fax: q1-216-368-5824; E-mail: [email protected]
epithelial cells and vascular endothelial cells comprising the blood–retinal barrier w19,32x. Relatively low levels of Glut3 is expressed in the retina being localized to the plexiform layer and vascular endothelial cells w17,35x. Stimulation of Glut1 gene expression in response to hypoxia and or to inhibition of oxidative phosphorylation has been studied in some detail in model cell culture systems, including aortic endothelial cells, 3T3-L1 adipocytes, L6 myocytes, and several liver-derived cell lines w2,3,13,21,22,27,28x. Results of studies in the Clone 9 rat liver cell line indicate that Glut1 mRNA content is increased in response to inhibition of oxidative phosphorylation, and that the effect is mediated by enhanced Glut1 gene transcription as well as decreased Glut1 mRNA degradation w27x. It has also been shown that chronic exposure of rats to hypobaric hypoxia Ž10% O 2 for 1 to 3 weeks. stimulates the expression of Glut1 in brain microvessels, a change that is associated with a 60% increase in the capillary density of the cerebrum, and is accompa-
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 3 0 1 - 5
G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
nied by an increase in the capacity of the brain to transport glucose w10,12x. Glut1 and Glut3 expression in brain is also augmented in response to ischemia in vivo w7,20,34x. Occlusion of the middle cerebral or carotid artery increased expression of both Glut1 and Glut3 isoforms w7,20,34x. Interestingly, Glut1 becomes transiently expressed in neuronal cells following ischemia w7,20x, and its expression is enhanced by chronic insulin-induced hypoglycemia w33x. In a previous study, we showed that the stimulation of Glut1 expression in response to hypoxia is mediated by two distinct pathways w3x. The first pathway involves oxygen-sensing regulatory molecules and hypoxia-inducible factor-1 ŽHIF-1. which mediate the enhanced expression of erythropoietin ŽEPO. and vascular endothelial growth factor ŽVEGF. genes in response to hypoxia w8,36x. The hypoxia-responsive pathway is also stimulated by exposure to cobalt chloride Žin the presence of oxygen. without inhibition of oxidative phosphorylation w3x. The second pathway leading to induction of Glut1 gene expression is activated in response to inhibition of oxidative phosphorylation w22,27,28x. This latter response is observed following use of agents that inhibit mitochondrial function such as sodium azide w21,27x. Indeed we have found that the transcriptional stimulation of Glut1 gene expression by cobalt chloride wCoŽII.x and azide are additive, and are mediated by different regions of the Glut1 promoter w3x. We have recently extended these studies focused on the regulation of Glut1 expression by cobalt chloride to in vivo models w40x. Addition of CoŽII. to the drinking water of rats resulted in a 1.3- to 2.5-fold increase in the content of Glut1 mRNA in cerebrum, kidney cortex, liver, skeletal muscle, and ventricular myocardium of both normal and diabetic rats w40x. However, whether the increase in the content of Glut1 mRNA results in higher levels of Glut1 expression was not determined. The present study was hence designed to address the following questions: Ž1. Does the increase in Glut1 mRNA expression in CoŽII.treated rats lead to increased Glut1 protein expression in cerebrum and retina?; Ž2. Is Glut3 expression also enhanced by treatment with CoŽII.?; Ž3. Is the presumed increase in the content of Glut1 in cerebrum and retina induced by CoŽII. associated with increased Glut1 content in microvasculature of these tissues?; and Ž4. Is treatment with CoŽII. associated with increased microvasculature density in brain and retina?
2. Materials and methods 2.1. Materials Nitrocellulose ŽBAS-85. was obtained from Schleicher and Schuell ŽKeene, NH.. Cobalt chloride, TRI REAGENT, and standard chemicals were obtained from Sigma ŽSt.
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Louis, MO.. Deoxycytidine 5X-Ž a-32 P. triphosphate Ž3000 Cirmmol. was obtained from DuPont-New England Nuclear ŽBoston, MA.. Protein assay kit was purchased from Bio-Rad ŽHercules, CA.. Affinity purified rabbit anti-Glut1 and anti-Glut3 IgG were obtained from Charles River Pharmaservices ŽSouthbridge, MA.. Rabbit anti-VEGF was purchased from Santa Cruz Biotechnology ŽSanta Cruz, CA.. Horseradish peroxidase-labeled Streptavidin-Biotin system was obtained from Dako ŽCarpenteria, CA.. Premium quality peroxidase-labeled goat anti-rabbit IgG ŽH & L. was obtained from Gibco BRL ŽGaithersburg, MD.. Enhanced chemiluminescence kit ŽECL. was purchased from Amersham Life Science ŽBuckinghamshire, England.. pSPT18 plasmid was from Boehringer Mannheim ŽIndianapolis, IN., and random hexamers, SP6 RNA polymerase, and restriction enzymes were obtained from Promega ŽMadison, WI.. Superscript II reverse transcriptase and Taq DNA polymerase were purchased from Gibco ŽGrand Island, NY.. DNA primers were from Oligos Etc. and Oligo Therapeutics ŽWilsonville, OR.. 2.2. Animal protocols Male Sprague–Dawley rats Ž225 to 250 g. were fed standard rat chow. Cobalt chloride was added to the drinking water Ž2 mM solution. for 10 to 12 days. Based on the daily water intake, the total dose of CoŽII. was estimated to be 0.5 to 0.6 mmol per animal. Rats were sacrificed at the end of the above treatment by CO 2 anesthesia followed by decapitation. Brain was isolated and the cerebellum was dissected away. Cerebral gray matter was used for isolation of microvessels, and samples from the frontal lobe and hippocampus were frozen at y808C for immunohistochemistry. Eyes Žcomplete globes. were either used for isolation of retinal microvasculature, or wrapped in aluminum foil and frozen on dry ice and stored at y808C. Following thawing of frozen globes, retinae were isolated by sharp dissection and immediately re-frozen until use. All assays were performed within 2 months of sacrifice. 2.3. Isolation of cerebral RNA and Northern blot analysis Fifty milligrams of brain tissue Žobtained from the gray matter of the frontal lobe. was thawed in 1.0 ml of TRI REAGENT and total RNA was isolated according to supplier’s protocol. RNA Ž20 mg per lane. was fractionated in 1% agarose-formaldehyde gels and transferred by capillary action to nitrocellulose paper. The resulting blots were probed sequentially with 20–50 = 10 6 c.p.m. of Glut1 and Glut3 cDNAs w4,14x. Both probes were 32 P-labeled by the random priming method. To ensure equal RNA loading of the lanes and complete transfer to nitrocellulose membranes, ethidium bromide staining of ribosomal 28 and 18S bands was monitored throughout. Relative Glut mRNA levels were determined by laser scanning densitometry. On each blot, the density of the specific Glut mRNA band in
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G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
RNA samples derived from control rats were averaged and set to 1.0. The density of the bands in lanes containing RNA from CoŽII.-treated animals were normalized against the mean of the control group. 2.4. Quantitation of Glut1 mRNA in retina by quantitatiÕe reÕerse transcriptase competitiÕe PCR Procedures described for competitive PCR w41x were adapted for analysis of Glut1 mRNA in retina. Full-length of rat Glut1 cDNA was cloned into the EcoRI site of pSPT18. The XhoI site which is located at q191 of the cDNA w4x was eliminated by digestion with XhoI followed by ‘filling-in’ with Klenow fragment DNA polymerase and ligation. Following digestion with BsteII Žposition q976., in-vitro transcription was performed employing SP6 RNA polymerase and the purity of the RNA product was verified by fractionation on SDS-urea gels. The resulting 976 base cRNA was used as internal control in the reverse transcription reaction. Total RNA was isolated from retina using TRI REAGENT as described above. Reverse transcription ŽRT. was performed using random hexamers. In each 20 ml reaction, 10 pg of the transcribed cRNA was added to 1.0 mg of retinal total RNA. 5 ml of the RT product was then used as template in the subsequent PCR reaction. Care was taken to ensure that all samples received the same amount of the cRNA. The upstream primer was from position q57 to q86 of rat Glut1 cDNA w4x and had the following sequence: 5X-ACTGGTACCAGAACACAAGAATCCCTTGTG. The downstream primer was from position q756 to q732 of Glut1 cDNA with the following sequence: 5X-GCATTGCCCATGATGGAGTCTAAGCG. Annealing and extension temperatures were 52 and 728C, respectively, and the PCR was performed for 35 cycles. PCR products Ž699 and 703 bp. were digested with XhoI. Because the rat Glut1 gene contains 4 introns and ; 24 kbp of DNA sequence between the location of the two primers w38x, any potential contamination of retinal RNA samples with genomic DNA would not be expected to result in a PCR product. The 699 bp PCR product derived from Glut1 mRNA from retina was restricted by XhoI into 565 and 134 bp fragments, while the 703 bp PCR product derived from the added cRNA was resistant to digestion by the enzyme. Products were fractionated in a 1.2% agarose gel and the intensity of ethidium bromide fluorescence of each band was determined using Image Analysis System ŽBio-Rad, Hercules, CA. and an appropriate computer program ŽMolecular Analyst, Promega, Madison, WI.. The ratio of the intensity of the 565 bp band to the 699 bp band was calculated for each sample of retina RNA, i.e., the ratio of DNA derived from retina Glut1 mRNA divided by that derived form Glut1 cRNA containing the modified XhoI site. The ratios derived from RNA isolated from retina of normal rats were averaged and set to 1.0. The calculated ratio from each RNA sample isolated from the
retina of CoŽII.-treated rats was normalized against the mean value derived from normal rats, and the results were averaged. 2.5. Preparation of protein samples from cerebrum and retina, and Western blot analysis Samples of brain Ž; 20 mg. obtained from gray matter of frontal lobe and from retina Ž; 7 mg. were homogenized at a 1:15 dilution Žweightrvolume. in a solution containing 0.25 M sucrose, 10 mM Tris, 0.5 mM EGTA, pH 7.4, for 10 strokes using a glass-teflon homogenizer. Homogenates were centrifuged at 2000 = g for 5 min and the resulting post-nuclear supernatants were used in Western blot analysis. For the cerebrum, 50 and 20 mg protein was loaded per lane in blots to be reacted with anti-Glut1 and anti-Glut3 antibodies, respectively. For the retina, 30 and 75 mg of protein was loaded per lane in blots for analysis of Glut1 and Glut3, respectively. Following SDSPAGE and transfer to nitrocellulose membrane, blots were incubated in Tris buffered saline Ž20 mM Tris and 137 mM NaCl, pH 7.6. containing 0.1% Tween 20 ŽTBS-T., and 5% nonfat milk. Following three washes each for 10 min in TBS-T, blots were incubated with 1:300 dilution of anti-Glut1 or 1:10,000 dilution of anti-Glut3 IgG in TBS-T for 1 h. After three washes with TBS-T, blots were reacted with 1:2000 dilution of peroxidase-labeled goat anti-rabbit IgG in TBS-T containing 0.15% nonfat milk for 1 h, washed 3 times with TBS devoid of the detergent, and developed using an ECL kit. 2.6. Isolation of brain microÕessels Published methods were followed with some modifications w29,31x. Briefly, brains from 3 rats were rapidly removed and immersed in ice-cold Earl’s-Hepes buffer containing Earl’s salts, 20 mM Hepes ŽpH 7.4., and 1% bovine serum albumin. The brainstem and cerebellum were dissected away and discarded, and the pia and other large superficial blood vessels were removed using fine forceps. The gray matter was isolated using a sharp razor blade and minced with a fine scissors. The small pieces of gray matter were homogenized by hand Ž3–4 strokes. in 30 ml of the above buffer using a 40 ml Dounce homogenizer with a loosely-fitting pestle. The homogenate was sieved through 210 mm nylon mesh and the material collected on the mesh was homogenized again in 20 ml of the buffer using 2–3 strokes. The homogenate was sieved through 105 mm nylon mesh and microvessels collected on the mesh were washed gently with 10 ml of the buffer, and collected. The material was layered over 5 ml of 1.3 M sucrose containing 10 mM Tris ŽpH 7.4., and centrifuged at 10,000 = g for 10 min at 48C. The pellet containing purified microvessels was washed twice with ice-cold PBS. The purity of each microvessel preparation was monitored by light microscopy and was similar in control and cobalt
G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
chloride-treated groups. We estimate that contaminants Žmostly naked nuclei. did not exceed 5% of the nuclei in the preparation, and the yield of microvessels from both groups was equal Ž; 80 mg protein.. A fraction of the isolated microvessels was used for immunohistochemistry Žsee below., and the remainder was used for Western blot analysis. Cerebral microvessels were solubilized in 1 = SDS sample buffer without bromphenol blue or glycerol and fragmented using a Sonicator disrupter ŽModel W 380. at the scale of 2 for 20–30 s. Protein was measured by Micro BCA reagent ŽPierce., and Western blots using 5 mg protein per sample were performed as described above. 2.7. Isolation of retinal microÕessels Recovery of microvessels from rat retina using the method employed for isolation of microvessels from cerebral cortex was poor. Thus, retinal vasculature was isolated from whole retina by the osmotic shock method, as described previously w18x. Briefly, freshly isolated retina were incubated in distilled water for 1 h, followed by a 5 min incubation with DNase I Ž2 mgrml.. Retinal microvasculature was isolated under microscopy by repetitive inspiration and ejection through Pasteur pipettes with sequentially narrower tips. Retinal microvessels isolated by this method showed a normal complement of nuclei and
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were devoid of all nonvascular materials. The vasculature of one eye per group of 3 rats was laid out on a glass slide and air-dried for immunohistochemistry, and the remaining microvasculature preparations Ž2–3. were frozen for Western blot analysis, as described above. Protein content was measured following sonication in 1 = SDS sample buffer as described above, and 5 mg of protein was used per lane. 2.8. Immunohistochemistry of cerebral gray matter and retina for Glut1 2.8.1. Whole tissue Immunostaining of tissue sections was performed on formalin-fixed paraffin embedded samples of cerebrum Žfrontal lobe and hippocampus. and retina employing avidin–biotin–peroxidase reagents. Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide. The slides were then subjected to an epitope enhancement procedure involving a 10 min irradiation in a 10 mM citrate buffer ŽpH 6.0. in an 850 W microwave. Primary anti-Glut1 IgG was diluted Ž1:200. in a background-reducing buffer ŽDako. and applied to tissue sections; slides were then incubated in a humidity chamber overnight at room temperature. The secondary antibody Žanti-rabbit immunoglobulin labeled with biotin. was applied, followed by washing and use of streptavidin-tagged
Fig. 1. Cobalt chloride increases Glut1 expression in cerebrum. ŽA. Post-nuclear cerebral gray matter homogenates from control and CoŽII.-treated rats were analyzed by Western blot using anti-Glut1 IgG. Blots were developed by an enhanced chemiluminescence kit ŽECL reagent.. Molecular mass markers ŽkDa. are shown on the left. Lanes 1 and 3 are from control rats; lanes 2 and 4 are from CoŽII.-treated rats. ŽB. Left Panel. Northern blots were prepared from total RNA isolated from cerebral gray matter of normal rats or rats treated for 10 days with CoŽII.. The blots were hybridized with rat Glut1 cDNA probe. The experiment was repeated twice with 6 rats per group. In each experiment, Glut1 mRNA content in CoŽII.-treated rats was normalized against the mean of the control group and the results were averaged and are expressed as mean" S.E.M. Ž n s 12; ) P - 0.05.. Right Panel. Post-nuclear homogenates were fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and reacted with anti-Glut1 IgG. The experiment was performed twice with 6 rats in each group Ž n s 12; ) P - 0.05..
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G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
with horseradish peroxidase. Visualization of the bound antigen was accomplished by addition of 3-3,diaminobenzidine ŽDAB. and hydrogen peroxide. Slides were counter-stained briefly in Harris hematoxylin prior to placement of coverslips. 2.8.2. Isolated microÕessels Immunostaining of isolated microvessels was performed with some modifications of the above procedure. Briefly, microvessels were air-dried onto glass microscope slides and then fixed in 100% methanol at y208C for 5 min. After washing three times with PBS, cells were permeabilized with 0.05% Tween 20 in PBS for 20 min, washed 3 times with PBS, and reacted for 1 h with rabbit anti-Glut1 IgG at 1:50 dilution in PBS containing 0.2% goat serum for retina and 1:100 dilution for brain. Slides were washed three times with PBS and incubated with goat anti-rabbit
IgG-labeled with peroxidase at the dilutions mentioned above for 1 h. After washing 3 times with 0.05% Tween 20 in PBS Žeach for 5 min., slides were incubated with ImmunoPure Metal Enhanced DAB Substrate ŽPierce. for 5 min, washed with PBS and mounted using an aqueousbased mounting medium. The intensity of Glut1 staining in isolated retinal and brain cortical microvessels was measured independently on a scale of 0 to 3 in a blinded fashion by two observers. Scores given to a minimum of 10 microvessels in each preparation derived from control and CoŽII.-treated rats were averaged. 2.9. Estimation of microÕessels density in brain and retina The density of microvessels in retina and in gray matter of frontal lobe and hippocampus was quantitated in paraf-
Fig. 2. Effect of CoŽII. on cerebral Glut3 mRNA and Glut3 protein content. ŽA. Post-nuclear cerebral gray matter homogenates from control and CoŽII.-treated rats were analyzed by Western blot using anti-Glut3 IgG. Molecular mass markers are shown on the left side. Lanes 1, 3, 5, 7, and 9 are from control rats while lanes 2, 4, 6, 8, and 10 are from CoŽII.-treated rats. ŽB. Left Panel. Northern blot analysis was performed on total RNA. The resulting blots were probed with human Glut3 cDNA. The experiment was repeated twice with 4 rats in each group, and the results were averaged; n s 8; means" S.E.M. The change in CoŽII.-treated rats is not significant. Right Panel. Western blots of the cerebral tissue were reacted with anti-Glut3 IgG. The experiment was repeated twice and the results averaged Ž n s 10; ) P - 0.05..
G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
fin sections using point-counting methods w6x. Hematoxylin and eosin-stained sections were examined at 250 = , and the fraction of grid points Ž10 = 10 grid. intersecting with capillary cross-sections counted. For frontal lobe and hippocampus, the density of capillary profiles was calculated by averaging four different regions per tissue section Žtotal area of 0.67 mm2 .. The density of capillaries in retinal cross-sections were estimated as the number of capillary profiles observed per 40 mm linear length of retina. Because the number of microvessels counted in retinal crosssections was modest, the density of retinal microvessels was also assessed in Periodic acid-Schiff-stained flat preparations of isolated retinal vasculature. The two methods employed for measurement of retinal vascular density yielded similar results. 2.10. Statistical analysis Results are expressed as means " S.E.M. Students unpaired two-tailed t-test was used and a P - 0.05 was considered significant w30x.
3. Results Animals treated with CoŽII. for 10–12 days showed no gross abnormality and no significant change in their fasting blood glucose levels, as noted previously w40x. Hemat-
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ocrit increased from 45.0 " 0.3 in controls to 47.3 " 0.3% in CoŽII.-treated rats Ž P - 0.05.. The effect of CoŽII. on Glut1 expression was measured at the level of mRNA and protein by quantitative Northern and Western blot analysis, respectively. We first tested the efficacy of anti-Glut1 IgG to detect Glut1 in cerebral homogenates ŽFig. 1A.. Glut1 protein migrated as a broad band ranging from ; 45 to ; 55 kDa, as described previously w24,28x. Treatment of rats with CoŽII. appeared to increase the intensity of the Glut1 band, while no qualitative change in its migration pattern was observed. We have previously employed the Northern blot technique for the quantitation of Glut1 mRNA in brain and other tissue w27,28,40x, and applied similar techniques in the present studies. To determine the effect of CoŽII. on Glut1 expression in the cerebrum, the relative abundance of Glut1 mRNA and Glut1 protein in cerebral gray matter Žfrontal lobe. of control and CoŽII.-treated rats was measured. Treatment with CoŽII. resulted in a significant increase Ž1.45-fold. in the content of Glut1 mRNA in comparison to normal controls Ž n s 12; P - 0.05. ŽFig. 1B, left panel.. The increase in the content of Glut1 mRNA was associated with a 1.4-fold increase in the content of Glut1 protein ŽFig. 1B, right panel; P - 0.05.. Fig. 2A demonstrates Glut3 expression in cerebrum of control and CoŽII.-treated rats. Glut3 migrated as a predominant band at ; 45 kDa; a minor band migrating at ; 58 kDa was also present and became more evident in
Fig. 3. Cobalt chloride increases the content of Glut1 mRNA and Glut1 protein in rat retina. Left Panel. Glut1 mRNA content in RNA derived from retina of CoŽII.-treated rats was calculated as the ratio to the average Glut1 mRNA content in RNA derived from retina of normal rats Ž n s 4 per group; ) P - 0.05.. Right Panel. Post-nuclear homogenates prepared from retina were fractionated by SDS-PAGE, and transferred to nitrocellulose membrane. Blots were reacted with anti-Glut1 IgG. The experiment was performed twice with 6 rats in each group Ž n s 12; ) P - 0.05..
G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
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Table 1 Effect of treatment with CoŽII. on Glut1 expression in microvessels isolated from cerebral gray matter and retina Method
Cerebral microvessels Control
CoŽII.
Retinal microvessels Control
CoŽII.
Western blot 1.00"0.10 1.54"0.17) 1.00".08 1.35"0.10) Immunohisto- 1.06"0.30 2.30"0.14) 1.12"0.10 1.68"0.25) chemistry ns 7, mean"S.E.M. )Denotes P - 0.05 as compared to control.
CoŽII.-treated animals. The upper band was not present in samples derived from retina Žnot shown.. The effect of treatment with CoŽII. on the content of Glut3 mRNA and Glut3 protein in cerebral gray matter was quantitated ŽFig. 2B.. In contrast to Glut1 mRNA, the content of Glut3 mRNA was not changed in CoŽII.-treated rats ŽFig. 2B, left panel.; this result is consistent with our previous findings w40x. Interestingly, and despite the lack of increase in the abundance of Glut3 mRNA, the relative abundance of Glut3 protein was significantly increased in cerebral tissue of CoŽII.-treated animals ŽFig. 2B, right panel; P - 0.05.. To extend the above analysis to retina, we first determined the relative abundance of Glut1 and Glut3 in the cerebrum as compared to retina in control rats. Equal amounts of post-nuclear homogenate protein derived from four retina and five samples of cerebral gray matter were fractionated and the resulting blots were reacted with anti-Glut1 and anti-Glut3 IgGs. Relative Glut1 abundance
was 6 " 1.5 times higher in retina than in brain, whereas Glut3 was 12 " 2-fold greater in brain than in retina. The potential effect of CoŽII. on Glut1 and Glut3 expression in the retina was next determined. Treatment with CoŽII. resulted in 2.5- and 1.23-fold increases in the content of Glut1 mRNA and Glut1 protein in retina, respectively ŽFig. 3; P - 0.05 for both changes.. In addition, there was a significant 1.5 " 0.1-fold increase in the content of Glut3 in retina of CoŽII.-treated rats. The possibility that the increase in Glut1 expression observed in the cerebral gray matter and retina is reflected in their respective microvasculature was explored. To perform these studies, microvessels from cerebral gray matter and retina of control and CoŽII.-treated rats were isolated. Glut1 expression in cerebral and retinal microvessels was assayed by Western blotting as well as by immunohistochemistry ŽTable 1.. Treatment with CoŽII. resulted in a significant increase in the expression of Glut1 protein in the microvasculature of both tissues employing either Western blot analysis or immunohistochemistry. We also determined the effect of treatment with CoŽII. on expression of VEGF in cerebral gray matter by Western blot analysis. The abundance of the ; 14 kDa monomeric VEGF protein was significantly increased in cerebrum of CoŽII.-treated rats Ž1.0 " 0.1 vs. 2.2 " 0.3 in control and CoŽII.-treated rats, respectively; P - 0.05.. The effect of CoŽII. on VEGF expression in the retina was not determined. Exposure of rats to hypobaric hypoxia for 1 week has been reported to increase Glut1 expression in cerebral
Fig. 4. Lack of effect of CoŽII. on capillary density of cerebral gray matter and retina. Paraffin cross-sections of the frontal lobe, hippocampus, and retina from control Žstippled bars. and CoŽII.-treated rats Žsolid bars. were stained with hematoxylin and eosin. The density of cerebral microvessels was quantitated in a total area of 0.67 mm2 , whereas retinal microvessel density was estimated in 40 mm linear length of retina Ž n s 7; means" S.E.M...
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microvasculature, an effect that is associated with increased capillary density w11x. We hence explored the possibility that treatment with CoŽII. likewise leads to increased microvascularity in cerebrum and retina, perhaps resulting from increased expression of VEGF induced by CoŽII.. Capillary density measured in hippocampus and frontal lobe, as well as in retina, was found to be unchanged in rats treated with CoŽII. for a period of 10–12 days ŽFig. 4..
4. Discussion Results of the present study show for the first time that treatment of rats with cobalt chloride, an agent which stimulates the expression of a family of hypoxia-responsive genes w8,36x, augments the expression of Glut1 and Glut3 in the brain and retina. The results also document that the increase in the content of Glut1 in cerebrum and retina is associated with increased Glut1 expression in their respective microvessels. These observations are consistent with previous reports demonstrating enhanced expression of Glut1 in the cerebral microvasculature in response to hypobaric hypoxia w7,10,12,20,34x. Moreover, the response to CoŽII. suggests that the stimulation of Glut1 expression by hypoxia may in large part be mediated by oxygen-sensing and signaling pathways that are operative in the responsive cells in these tissues. The available evidence indicates that Glut3 expressions is localized to neuronal cells of the cerebral cortex w14,24x. Results of present study show that the content of Glut3 is augmented in cerebral gray matter of CoŽII.-treated animals. Taken in conjunction with the increased Glut1 expression, the above findings suggest that increased function of the two transporters operating in series—with Glut1 mediating transport of glucose out of the microvasculature, and Glut3 mediating the cellular uptake of glucose—would result in higher rates of glucose uptake and metabolism by the cerebrum of CoŽII.-treated rats. However, unlike the regulation of Glut1 expression, the increase in Glut3 content was not associated with any change in the relative abundance of Glut3 mRNA in cerebrum, suggesting regulation at the translational or post-translational levels. This inference is consistent with a recent report in L6 cells demonstrating that the induction of Glut3 protein in response to inhibition of oxidative phosphorylation by dinitrophenol occurs in the absence of any change in the content of Glut3 mRNA, and is mediated by increased stability of Glut3 protein w15x. Treatment with CoŽII. resulted in significant increases in the contents of both Glut1 and Glut3 in retina. Glut3 is expressed at significantly lower levels in retina than is Glut1 w35x. Glut1, however, is expressed at very high levels in cells comprising the blood–retinal barrier w32x. In vascular endothelial cells, Glut1 is present at a higher density in the ablumenal as compared to the lumenal
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plasma membrane w19x. Because of Glut1’s localization and function, it has been proposed that alterations in the expression of this isoform may play a role in the pathogenesis of retinal complications of diabetes w16,19x. Results of the present study demonstrate that the increase in the content of Glut1 in retina is associated with increased Glut1 expression in its microvasculature. This latter finding suggests that exposure of retina to hypoxia in the presence of elevated blood glucose concentrations Žsuch as in diabetes. may well lead to large increases in the amount of glucose transported into this tissue. Previous reports indicate that the increase in Glut1 expression in cerebral microvessels in rats exposed to hypobaric hypoxia is associated with increased cerebral capillary density w10–12x. The increase in microvascularity is near-maximal at 1 week of exposure w11x and primarily reflects an increase in the length, rather than in the number, of capillaries w23x. The available evidence also indicates that cobalt chloride, acting in the presence of oxygen and without inhibiting oxidative phosphorylation, acts as a ‘surrogate’ of hypoxia and leads to enhanced expression of a set of hypoxia-responsive-genes, including the gene encoding VEGF and Glut1 w3,8,37x; this effect is mediated by activation of HIF-1, a basic-helix-loop-helix-PAS transcription factor w36,37x. Considerable evidence indicates that VEGF plays a critical role in the development of neovascularization w26,39x, but there is conflicting data as to whether VEGF alone is sufficient to produce new vessel growth. Both patients w1x and animals w25x with early diabetic retinopathy or other retinal diseases have elevated levels of retinal VEGF, without any evidence of neovascularization. Results of the present study indicate that although treatment with CoŽII. for 10–12 days increased the hematocrit, and stimulated the expression of Glut1 in cerebrum and retina and VEGF in cerebrum, the treatment did not change microvascular density in either tissue. This finding suggests that the presumed CoŽII.-induced activation of HIF-1, and the resulting increase in VEGF expression, while necessary, may not be sufficient to induce hypervascularity in the tissues examined. Whether or not the concentration of VEGF achieved a level sufficient to increase vascular density remains unclear. Alternatively, it is possible that unknown tissue factors that are released or activated secondary to inhibition of oxidative phosphorylation by hypoxia play an important role in increasing the microvascular density. Further studies are necessary to clarify the roles of inhibition of oxidative phosphorylation and stimulation of hypoxia-signaling pathway in the overall adaptive response to hypoxia.
Acknowledgements We are grateful to the late Dr. Ora M. Rosen of the Memorial Sloan-Kettering Cancer Center for supplying full-length rat GLUT1 cDNA. Human GLUT3 cDNA was
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kindly provided by Dr. Graeme I. Bell of the Howard Hughes Medical Institute at the University of Chicago. This study was supported in part by grants from the Diabetes Association of Greater Cleveland, and by National Institutes of Health aDK45945 to F. Ismail-Beigi, and aEY003000 to T.S. Kern. Dr. Badr was supported by a grant from the Government of Egypt and Dr. J. Tang is a recipient of a fellowship from Lions Club International Sightfirst program administered by the American Diabetes Association.
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Research report
Glut1 and Glut3 expression, but not capillary density, is increased by cobalt chloride in rat cerebrum and retina Gamal A. Badr, Jin-Zhong Zhang, Jie Tang, Timothy S. Kern, Faramarz Ismail-Beigi
)
Departments of Medicine and Physiology and Biophysics, and Diabetes Research Center, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106-4951, USA Accepted 27 October 1998
Abstract Treatment of rats with cobalt chloride wCoŽII.x, an agent that stimulates the expression of a set of hypoxia-responsive genes, for 10–12 days resulted in 1.45- and 1.40-fold increases in the content of Glut1 mRNA and Glut1 in cerebral gray matter, respectively Ž P - 0.05 for both changes.. The increase in Glut1 content was associated with a significant increase in the content of Glut1 staining in microvessels isolated from cerebral gray matter, and in the intensity of Glut1 in microvessels of the frontal lobe and hippocampus assessed by immunohistochemistry. The abundance of Glut3 in cerebrum of CoŽII.-treated rats also increased by 1.3-fold Ž P - 0.05., but the increase was not associated with a change in the content of Glut3 mRNA. In retina, treatment with CoŽII. resulted in 2.48- and 1.23-fold increases in the content of Glut1 mRNA and Glut1 protein, respectively Ž P - 0.05 for both changes.; similar increases in Glut1 protein expression were observed in isolated retinal microvasculature. The content of Glut3 in retina also increased 1.5-fold in CoŽII.-treated rats Ž P - 0.05.. In addition, treatment with CoŽII. resulted in a significant 2.2-fold increase in the expression of VEGF in the cerebrum. However, despite the CoŽII.-induced increase in Glut1 expression in cerebral and retinal microvasculature and VEGF in cerebrum, there was no increase in the capillary density in either tissue. It is concluded that a 10–12 day exposure to CoŽII., presumably acting through the hypoxia-signaling pathway, results in enhanced expression of both major glucose transporters in cerebral cortex and retina, without increasing the capillary density of either tissue. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Blood–brain barrier; Blood–retinal barrier; Glut1 mRNA; Glut3 mRNA; Competitive PCR; Brain microvessel; Retinal microvasculature; Capillary density; Hypoxia; VEGF
1. Introduction The metabolic activity of the central nervous system and the retina is critically dependent on metabolism of glucose as the predominant energy substrate w5,13x. Glucose transport across plasma membranes is mediated by a family of six sodium-independent glucose transporter glycoprotein molecules ŽGluts., which manifest distinct kinetic properties and tissue-specific patterns of expression w24x. In brain, Glut1 is highly expressed in endothelial cells of the blood–brain barrier ŽBBB. and to the choroid plexus epithelial cells w12,17,34x. Glut3, the other major Glut isoform present in the brain, is expressed at very high levels in neuronal cells, but not in brain microvessels w9,14,24x. In retina, Glut1 is highly expressed in pigment ) Corresponding author. Clinical and Molecular Endocrinology, Case Western Reserve University, Cleveland, OH 44106-4951, USA. Fax: q1-216-368-5824; E-mail: [email protected]
epithelial cells and vascular endothelial cells comprising the blood–retinal barrier w19,32x. Relatively low levels of Glut3 is expressed in the retina being localized to the plexiform layer and vascular endothelial cells w17,35x. Stimulation of Glut1 gene expression in response to hypoxia and or to inhibition of oxidative phosphorylation has been studied in some detail in model cell culture systems, including aortic endothelial cells, 3T3-L1 adipocytes, L6 myocytes, and several liver-derived cell lines w2,3,13,21,22,27,28x. Results of studies in the Clone 9 rat liver cell line indicate that Glut1 mRNA content is increased in response to inhibition of oxidative phosphorylation, and that the effect is mediated by enhanced Glut1 gene transcription as well as decreased Glut1 mRNA degradation w27x. It has also been shown that chronic exposure of rats to hypobaric hypoxia Ž10% O 2 for 1 to 3 weeks. stimulates the expression of Glut1 in brain microvessels, a change that is associated with a 60% increase in the capillary density of the cerebrum, and is accompa-
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 3 0 1 - 5
G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
nied by an increase in the capacity of the brain to transport glucose w10,12x. Glut1 and Glut3 expression in brain is also augmented in response to ischemia in vivo w7,20,34x. Occlusion of the middle cerebral or carotid artery increased expression of both Glut1 and Glut3 isoforms w7,20,34x. Interestingly, Glut1 becomes transiently expressed in neuronal cells following ischemia w7,20x, and its expression is enhanced by chronic insulin-induced hypoglycemia w33x. In a previous study, we showed that the stimulation of Glut1 expression in response to hypoxia is mediated by two distinct pathways w3x. The first pathway involves oxygen-sensing regulatory molecules and hypoxia-inducible factor-1 ŽHIF-1. which mediate the enhanced expression of erythropoietin ŽEPO. and vascular endothelial growth factor ŽVEGF. genes in response to hypoxia w8,36x. The hypoxia-responsive pathway is also stimulated by exposure to cobalt chloride Žin the presence of oxygen. without inhibition of oxidative phosphorylation w3x. The second pathway leading to induction of Glut1 gene expression is activated in response to inhibition of oxidative phosphorylation w22,27,28x. This latter response is observed following use of agents that inhibit mitochondrial function such as sodium azide w21,27x. Indeed we have found that the transcriptional stimulation of Glut1 gene expression by cobalt chloride wCoŽII.x and azide are additive, and are mediated by different regions of the Glut1 promoter w3x. We have recently extended these studies focused on the regulation of Glut1 expression by cobalt chloride to in vivo models w40x. Addition of CoŽII. to the drinking water of rats resulted in a 1.3- to 2.5-fold increase in the content of Glut1 mRNA in cerebrum, kidney cortex, liver, skeletal muscle, and ventricular myocardium of both normal and diabetic rats w40x. However, whether the increase in the content of Glut1 mRNA results in higher levels of Glut1 expression was not determined. The present study was hence designed to address the following questions: Ž1. Does the increase in Glut1 mRNA expression in CoŽII.treated rats lead to increased Glut1 protein expression in cerebrum and retina?; Ž2. Is Glut3 expression also enhanced by treatment with CoŽII.?; Ž3. Is the presumed increase in the content of Glut1 in cerebrum and retina induced by CoŽII. associated with increased Glut1 content in microvasculature of these tissues?; and Ž4. Is treatment with CoŽII. associated with increased microvasculature density in brain and retina?
2. Materials and methods 2.1. Materials Nitrocellulose ŽBAS-85. was obtained from Schleicher and Schuell ŽKeene, NH.. Cobalt chloride, TRI REAGENT, and standard chemicals were obtained from Sigma ŽSt.
25
Louis, MO.. Deoxycytidine 5X-Ž a-32 P. triphosphate Ž3000 Cirmmol. was obtained from DuPont-New England Nuclear ŽBoston, MA.. Protein assay kit was purchased from Bio-Rad ŽHercules, CA.. Affinity purified rabbit anti-Glut1 and anti-Glut3 IgG were obtained from Charles River Pharmaservices ŽSouthbridge, MA.. Rabbit anti-VEGF was purchased from Santa Cruz Biotechnology ŽSanta Cruz, CA.. Horseradish peroxidase-labeled Streptavidin-Biotin system was obtained from Dako ŽCarpenteria, CA.. Premium quality peroxidase-labeled goat anti-rabbit IgG ŽH & L. was obtained from Gibco BRL ŽGaithersburg, MD.. Enhanced chemiluminescence kit ŽECL. was purchased from Amersham Life Science ŽBuckinghamshire, England.. pSPT18 plasmid was from Boehringer Mannheim ŽIndianapolis, IN., and random hexamers, SP6 RNA polymerase, and restriction enzymes were obtained from Promega ŽMadison, WI.. Superscript II reverse transcriptase and Taq DNA polymerase were purchased from Gibco ŽGrand Island, NY.. DNA primers were from Oligos Etc. and Oligo Therapeutics ŽWilsonville, OR.. 2.2. Animal protocols Male Sprague–Dawley rats Ž225 to 250 g. were fed standard rat chow. Cobalt chloride was added to the drinking water Ž2 mM solution. for 10 to 12 days. Based on the daily water intake, the total dose of CoŽII. was estimated to be 0.5 to 0.6 mmol per animal. Rats were sacrificed at the end of the above treatment by CO 2 anesthesia followed by decapitation. Brain was isolated and the cerebellum was dissected away. Cerebral gray matter was used for isolation of microvessels, and samples from the frontal lobe and hippocampus were frozen at y808C for immunohistochemistry. Eyes Žcomplete globes. were either used for isolation of retinal microvasculature, or wrapped in aluminum foil and frozen on dry ice and stored at y808C. Following thawing of frozen globes, retinae were isolated by sharp dissection and immediately re-frozen until use. All assays were performed within 2 months of sacrifice. 2.3. Isolation of cerebral RNA and Northern blot analysis Fifty milligrams of brain tissue Žobtained from the gray matter of the frontal lobe. was thawed in 1.0 ml of TRI REAGENT and total RNA was isolated according to supplier’s protocol. RNA Ž20 mg per lane. was fractionated in 1% agarose-formaldehyde gels and transferred by capillary action to nitrocellulose paper. The resulting blots were probed sequentially with 20–50 = 10 6 c.p.m. of Glut1 and Glut3 cDNAs w4,14x. Both probes were 32 P-labeled by the random priming method. To ensure equal RNA loading of the lanes and complete transfer to nitrocellulose membranes, ethidium bromide staining of ribosomal 28 and 18S bands was monitored throughout. Relative Glut mRNA levels were determined by laser scanning densitometry. On each blot, the density of the specific Glut mRNA band in
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RNA samples derived from control rats were averaged and set to 1.0. The density of the bands in lanes containing RNA from CoŽII.-treated animals were normalized against the mean of the control group. 2.4. Quantitation of Glut1 mRNA in retina by quantitatiÕe reÕerse transcriptase competitiÕe PCR Procedures described for competitive PCR w41x were adapted for analysis of Glut1 mRNA in retina. Full-length of rat Glut1 cDNA was cloned into the EcoRI site of pSPT18. The XhoI site which is located at q191 of the cDNA w4x was eliminated by digestion with XhoI followed by ‘filling-in’ with Klenow fragment DNA polymerase and ligation. Following digestion with BsteII Žposition q976., in-vitro transcription was performed employing SP6 RNA polymerase and the purity of the RNA product was verified by fractionation on SDS-urea gels. The resulting 976 base cRNA was used as internal control in the reverse transcription reaction. Total RNA was isolated from retina using TRI REAGENT as described above. Reverse transcription ŽRT. was performed using random hexamers. In each 20 ml reaction, 10 pg of the transcribed cRNA was added to 1.0 mg of retinal total RNA. 5 ml of the RT product was then used as template in the subsequent PCR reaction. Care was taken to ensure that all samples received the same amount of the cRNA. The upstream primer was from position q57 to q86 of rat Glut1 cDNA w4x and had the following sequence: 5X-ACTGGTACCAGAACACAAGAATCCCTTGTG. The downstream primer was from position q756 to q732 of Glut1 cDNA with the following sequence: 5X-GCATTGCCCATGATGGAGTCTAAGCG. Annealing and extension temperatures were 52 and 728C, respectively, and the PCR was performed for 35 cycles. PCR products Ž699 and 703 bp. were digested with XhoI. Because the rat Glut1 gene contains 4 introns and ; 24 kbp of DNA sequence between the location of the two primers w38x, any potential contamination of retinal RNA samples with genomic DNA would not be expected to result in a PCR product. The 699 bp PCR product derived from Glut1 mRNA from retina was restricted by XhoI into 565 and 134 bp fragments, while the 703 bp PCR product derived from the added cRNA was resistant to digestion by the enzyme. Products were fractionated in a 1.2% agarose gel and the intensity of ethidium bromide fluorescence of each band was determined using Image Analysis System ŽBio-Rad, Hercules, CA. and an appropriate computer program ŽMolecular Analyst, Promega, Madison, WI.. The ratio of the intensity of the 565 bp band to the 699 bp band was calculated for each sample of retina RNA, i.e., the ratio of DNA derived from retina Glut1 mRNA divided by that derived form Glut1 cRNA containing the modified XhoI site. The ratios derived from RNA isolated from retina of normal rats were averaged and set to 1.0. The calculated ratio from each RNA sample isolated from the
retina of CoŽII.-treated rats was normalized against the mean value derived from normal rats, and the results were averaged. 2.5. Preparation of protein samples from cerebrum and retina, and Western blot analysis Samples of brain Ž; 20 mg. obtained from gray matter of frontal lobe and from retina Ž; 7 mg. were homogenized at a 1:15 dilution Žweightrvolume. in a solution containing 0.25 M sucrose, 10 mM Tris, 0.5 mM EGTA, pH 7.4, for 10 strokes using a glass-teflon homogenizer. Homogenates were centrifuged at 2000 = g for 5 min and the resulting post-nuclear supernatants were used in Western blot analysis. For the cerebrum, 50 and 20 mg protein was loaded per lane in blots to be reacted with anti-Glut1 and anti-Glut3 antibodies, respectively. For the retina, 30 and 75 mg of protein was loaded per lane in blots for analysis of Glut1 and Glut3, respectively. Following SDSPAGE and transfer to nitrocellulose membrane, blots were incubated in Tris buffered saline Ž20 mM Tris and 137 mM NaCl, pH 7.6. containing 0.1% Tween 20 ŽTBS-T., and 5% nonfat milk. Following three washes each for 10 min in TBS-T, blots were incubated with 1:300 dilution of anti-Glut1 or 1:10,000 dilution of anti-Glut3 IgG in TBS-T for 1 h. After three washes with TBS-T, blots were reacted with 1:2000 dilution of peroxidase-labeled goat anti-rabbit IgG in TBS-T containing 0.15% nonfat milk for 1 h, washed 3 times with TBS devoid of the detergent, and developed using an ECL kit. 2.6. Isolation of brain microÕessels Published methods were followed with some modifications w29,31x. Briefly, brains from 3 rats were rapidly removed and immersed in ice-cold Earl’s-Hepes buffer containing Earl’s salts, 20 mM Hepes ŽpH 7.4., and 1% bovine serum albumin. The brainstem and cerebellum were dissected away and discarded, and the pia and other large superficial blood vessels were removed using fine forceps. The gray matter was isolated using a sharp razor blade and minced with a fine scissors. The small pieces of gray matter were homogenized by hand Ž3–4 strokes. in 30 ml of the above buffer using a 40 ml Dounce homogenizer with a loosely-fitting pestle. The homogenate was sieved through 210 mm nylon mesh and the material collected on the mesh was homogenized again in 20 ml of the buffer using 2–3 strokes. The homogenate was sieved through 105 mm nylon mesh and microvessels collected on the mesh were washed gently with 10 ml of the buffer, and collected. The material was layered over 5 ml of 1.3 M sucrose containing 10 mM Tris ŽpH 7.4., and centrifuged at 10,000 = g for 10 min at 48C. The pellet containing purified microvessels was washed twice with ice-cold PBS. The purity of each microvessel preparation was monitored by light microscopy and was similar in control and cobalt
G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
chloride-treated groups. We estimate that contaminants Žmostly naked nuclei. did not exceed 5% of the nuclei in the preparation, and the yield of microvessels from both groups was equal Ž; 80 mg protein.. A fraction of the isolated microvessels was used for immunohistochemistry Žsee below., and the remainder was used for Western blot analysis. Cerebral microvessels were solubilized in 1 = SDS sample buffer without bromphenol blue or glycerol and fragmented using a Sonicator disrupter ŽModel W 380. at the scale of 2 for 20–30 s. Protein was measured by Micro BCA reagent ŽPierce., and Western blots using 5 mg protein per sample were performed as described above. 2.7. Isolation of retinal microÕessels Recovery of microvessels from rat retina using the method employed for isolation of microvessels from cerebral cortex was poor. Thus, retinal vasculature was isolated from whole retina by the osmotic shock method, as described previously w18x. Briefly, freshly isolated retina were incubated in distilled water for 1 h, followed by a 5 min incubation with DNase I Ž2 mgrml.. Retinal microvasculature was isolated under microscopy by repetitive inspiration and ejection through Pasteur pipettes with sequentially narrower tips. Retinal microvessels isolated by this method showed a normal complement of nuclei and
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were devoid of all nonvascular materials. The vasculature of one eye per group of 3 rats was laid out on a glass slide and air-dried for immunohistochemistry, and the remaining microvasculature preparations Ž2–3. were frozen for Western blot analysis, as described above. Protein content was measured following sonication in 1 = SDS sample buffer as described above, and 5 mg of protein was used per lane. 2.8. Immunohistochemistry of cerebral gray matter and retina for Glut1 2.8.1. Whole tissue Immunostaining of tissue sections was performed on formalin-fixed paraffin embedded samples of cerebrum Žfrontal lobe and hippocampus. and retina employing avidin–biotin–peroxidase reagents. Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide. The slides were then subjected to an epitope enhancement procedure involving a 10 min irradiation in a 10 mM citrate buffer ŽpH 6.0. in an 850 W microwave. Primary anti-Glut1 IgG was diluted Ž1:200. in a background-reducing buffer ŽDako. and applied to tissue sections; slides were then incubated in a humidity chamber overnight at room temperature. The secondary antibody Žanti-rabbit immunoglobulin labeled with biotin. was applied, followed by washing and use of streptavidin-tagged
Fig. 1. Cobalt chloride increases Glut1 expression in cerebrum. ŽA. Post-nuclear cerebral gray matter homogenates from control and CoŽII.-treated rats were analyzed by Western blot using anti-Glut1 IgG. Blots were developed by an enhanced chemiluminescence kit ŽECL reagent.. Molecular mass markers ŽkDa. are shown on the left. Lanes 1 and 3 are from control rats; lanes 2 and 4 are from CoŽII.-treated rats. ŽB. Left Panel. Northern blots were prepared from total RNA isolated from cerebral gray matter of normal rats or rats treated for 10 days with CoŽII.. The blots were hybridized with rat Glut1 cDNA probe. The experiment was repeated twice with 6 rats per group. In each experiment, Glut1 mRNA content in CoŽII.-treated rats was normalized against the mean of the control group and the results were averaged and are expressed as mean" S.E.M. Ž n s 12; ) P - 0.05.. Right Panel. Post-nuclear homogenates were fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and reacted with anti-Glut1 IgG. The experiment was performed twice with 6 rats in each group Ž n s 12; ) P - 0.05..
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with horseradish peroxidase. Visualization of the bound antigen was accomplished by addition of 3-3,diaminobenzidine ŽDAB. and hydrogen peroxide. Slides were counter-stained briefly in Harris hematoxylin prior to placement of coverslips. 2.8.2. Isolated microÕessels Immunostaining of isolated microvessels was performed with some modifications of the above procedure. Briefly, microvessels were air-dried onto glass microscope slides and then fixed in 100% methanol at y208C for 5 min. After washing three times with PBS, cells were permeabilized with 0.05% Tween 20 in PBS for 20 min, washed 3 times with PBS, and reacted for 1 h with rabbit anti-Glut1 IgG at 1:50 dilution in PBS containing 0.2% goat serum for retina and 1:100 dilution for brain. Slides were washed three times with PBS and incubated with goat anti-rabbit
IgG-labeled with peroxidase at the dilutions mentioned above for 1 h. After washing 3 times with 0.05% Tween 20 in PBS Žeach for 5 min., slides were incubated with ImmunoPure Metal Enhanced DAB Substrate ŽPierce. for 5 min, washed with PBS and mounted using an aqueousbased mounting medium. The intensity of Glut1 staining in isolated retinal and brain cortical microvessels was measured independently on a scale of 0 to 3 in a blinded fashion by two observers. Scores given to a minimum of 10 microvessels in each preparation derived from control and CoŽII.-treated rats were averaged. 2.9. Estimation of microÕessels density in brain and retina The density of microvessels in retina and in gray matter of frontal lobe and hippocampus was quantitated in paraf-
Fig. 2. Effect of CoŽII. on cerebral Glut3 mRNA and Glut3 protein content. ŽA. Post-nuclear cerebral gray matter homogenates from control and CoŽII.-treated rats were analyzed by Western blot using anti-Glut3 IgG. Molecular mass markers are shown on the left side. Lanes 1, 3, 5, 7, and 9 are from control rats while lanes 2, 4, 6, 8, and 10 are from CoŽII.-treated rats. ŽB. Left Panel. Northern blot analysis was performed on total RNA. The resulting blots were probed with human Glut3 cDNA. The experiment was repeated twice with 4 rats in each group, and the results were averaged; n s 8; means" S.E.M. The change in CoŽII.-treated rats is not significant. Right Panel. Western blots of the cerebral tissue were reacted with anti-Glut3 IgG. The experiment was repeated twice and the results averaged Ž n s 10; ) P - 0.05..
G.A. Badr et al.r Molecular Brain Research 64 (1999) 24–33
fin sections using point-counting methods w6x. Hematoxylin and eosin-stained sections were examined at 250 = , and the fraction of grid points Ž10 = 10 grid. intersecting with capillary cross-sections counted. For frontal lobe and hippocampus, the density of capillary profiles was calculated by averaging four different regions per tissue section Žtotal area of 0.67 mm2 .. The density of capillaries in retinal cross-sections were estimated as the number of capillary profiles observed per 40 mm linear length of retina. Because the number of microvessels counted in retinal crosssections was modest, the density of retinal microvessels was also assessed in Periodic acid-Schiff-stained flat preparations of isolated retinal vasculature. The two methods employed for measurement of retinal vascular density yielded similar results. 2.10. Statistical analysis Results are expressed as means " S.E.M. Students unpaired two-tailed t-test was used and a P - 0.05 was considered significant w30x.
3. Results Animals treated with CoŽII. for 10–12 days showed no gross abnormality and no significant change in their fasting blood glucose levels, as noted previously w40x. Hemat-
29
ocrit increased from 45.0 " 0.3 in controls to 47.3 " 0.3% in CoŽII.-treated rats Ž P - 0.05.. The effect of CoŽII. on Glut1 expression was measured at the level of mRNA and protein by quantitative Northern and Western blot analysis, respectively. We first tested the efficacy of anti-Glut1 IgG to detect Glut1 in cerebral homogenates ŽFig. 1A.. Glut1 protein migrated as a broad band ranging from ; 45 to ; 55 kDa, as described previously w24,28x. Treatment of rats with CoŽII. appeared to increase the intensity of the Glut1 band, while no qualitative change in its migration pattern was observed. We have previously employed the Northern blot technique for the quantitation of Glut1 mRNA in brain and other tissue w27,28,40x, and applied similar techniques in the present studies. To determine the effect of CoŽII. on Glut1 expression in the cerebrum, the relative abundance of Glut1 mRNA and Glut1 protein in cerebral gray matter Žfrontal lobe. of control and CoŽII.-treated rats was measured. Treatment with CoŽII. resulted in a significant increase Ž1.45-fold. in the content of Glut1 mRNA in comparison to normal controls Ž n s 12; P - 0.05. ŽFig. 1B, left panel.. The increase in the content of Glut1 mRNA was associated with a 1.4-fold increase in the content of Glut1 protein ŽFig. 1B, right panel; P - 0.05.. Fig. 2A demonstrates Glut3 expression in cerebrum of control and CoŽII.-treated rats. Glut3 migrated as a predominant band at ; 45 kDa; a minor band migrating at ; 58 kDa was also present and became more evident in
Fig. 3. Cobalt chloride increases the content of Glut1 mRNA and Glut1 protein in rat retina. Left Panel. Glut1 mRNA content in RNA derived from retina of CoŽII.-treated rats was calculated as the ratio to the average Glut1 mRNA content in RNA derived from retina of normal rats Ž n s 4 per group; ) P - 0.05.. Right Panel. Post-nuclear homogenates prepared from retina were fractionated by SDS-PAGE, and transferred to nitrocellulose membrane. Blots were reacted with anti-Glut1 IgG. The experiment was performed twice with 6 rats in each group Ž n s 12; ) P - 0.05..
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Table 1 Effect of treatment with CoŽII. on Glut1 expression in microvessels isolated from cerebral gray matter and retina Method
Cerebral microvessels Control
CoŽII.
Retinal microvessels Control
CoŽII.
Western blot 1.00"0.10 1.54"0.17) 1.00".08 1.35"0.10) Immunohisto- 1.06"0.30 2.30"0.14) 1.12"0.10 1.68"0.25) chemistry ns 7, mean"S.E.M. )Denotes P - 0.05 as compared to control.
CoŽII.-treated animals. The upper band was not present in samples derived from retina Žnot shown.. The effect of treatment with CoŽII. on the content of Glut3 mRNA and Glut3 protein in cerebral gray matter was quantitated ŽFig. 2B.. In contrast to Glut1 mRNA, the content of Glut3 mRNA was not changed in CoŽII.-treated rats ŽFig. 2B, left panel.; this result is consistent with our previous findings w40x. Interestingly, and despite the lack of increase in the abundance of Glut3 mRNA, the relative abundance of Glut3 protein was significantly increased in cerebral tissue of CoŽII.-treated animals ŽFig. 2B, right panel; P - 0.05.. To extend the above analysis to retina, we first determined the relative abundance of Glut1 and Glut3 in the cerebrum as compared to retina in control rats. Equal amounts of post-nuclear homogenate protein derived from four retina and five samples of cerebral gray matter were fractionated and the resulting blots were reacted with anti-Glut1 and anti-Glut3 IgGs. Relative Glut1 abundance
was 6 " 1.5 times higher in retina than in brain, whereas Glut3 was 12 " 2-fold greater in brain than in retina. The potential effect of CoŽII. on Glut1 and Glut3 expression in the retina was next determined. Treatment with CoŽII. resulted in 2.5- and 1.23-fold increases in the content of Glut1 mRNA and Glut1 protein in retina, respectively ŽFig. 3; P - 0.05 for both changes.. In addition, there was a significant 1.5 " 0.1-fold increase in the content of Glut3 in retina of CoŽII.-treated rats. The possibility that the increase in Glut1 expression observed in the cerebral gray matter and retina is reflected in their respective microvasculature was explored. To perform these studies, microvessels from cerebral gray matter and retina of control and CoŽII.-treated rats were isolated. Glut1 expression in cerebral and retinal microvessels was assayed by Western blotting as well as by immunohistochemistry ŽTable 1.. Treatment with CoŽII. resulted in a significant increase in the expression of Glut1 protein in the microvasculature of both tissues employing either Western blot analysis or immunohistochemistry. We also determined the effect of treatment with CoŽII. on expression of VEGF in cerebral gray matter by Western blot analysis. The abundance of the ; 14 kDa monomeric VEGF protein was significantly increased in cerebrum of CoŽII.-treated rats Ž1.0 " 0.1 vs. 2.2 " 0.3 in control and CoŽII.-treated rats, respectively; P - 0.05.. The effect of CoŽII. on VEGF expression in the retina was not determined. Exposure of rats to hypobaric hypoxia for 1 week has been reported to increase Glut1 expression in cerebral
Fig. 4. Lack of effect of CoŽII. on capillary density of cerebral gray matter and retina. Paraffin cross-sections of the frontal lobe, hippocampus, and retina from control Žstippled bars. and CoŽII.-treated rats Žsolid bars. were stained with hematoxylin and eosin. The density of cerebral microvessels was quantitated in a total area of 0.67 mm2 , whereas retinal microvessel density was estimated in 40 mm linear length of retina Ž n s 7; means" S.E.M...
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microvasculature, an effect that is associated with increased capillary density w11x. We hence explored the possibility that treatment with CoŽII. likewise leads to increased microvascularity in cerebrum and retina, perhaps resulting from increased expression of VEGF induced by CoŽII.. Capillary density measured in hippocampus and frontal lobe, as well as in retina, was found to be unchanged in rats treated with CoŽII. for a period of 10–12 days ŽFig. 4..
4. Discussion Results of the present study show for the first time that treatment of rats with cobalt chloride, an agent which stimulates the expression of a family of hypoxia-responsive genes w8,36x, augments the expression of Glut1 and Glut3 in the brain and retina. The results also document that the increase in the content of Glut1 in cerebrum and retina is associated with increased Glut1 expression in their respective microvessels. These observations are consistent with previous reports demonstrating enhanced expression of Glut1 in the cerebral microvasculature in response to hypobaric hypoxia w7,10,12,20,34x. Moreover, the response to CoŽII. suggests that the stimulation of Glut1 expression by hypoxia may in large part be mediated by oxygen-sensing and signaling pathways that are operative in the responsive cells in these tissues. The available evidence indicates that Glut3 expressions is localized to neuronal cells of the cerebral cortex w14,24x. Results of present study show that the content of Glut3 is augmented in cerebral gray matter of CoŽII.-treated animals. Taken in conjunction with the increased Glut1 expression, the above findings suggest that increased function of the two transporters operating in series—with Glut1 mediating transport of glucose out of the microvasculature, and Glut3 mediating the cellular uptake of glucose—would result in higher rates of glucose uptake and metabolism by the cerebrum of CoŽII.-treated rats. However, unlike the regulation of Glut1 expression, the increase in Glut3 content was not associated with any change in the relative abundance of Glut3 mRNA in cerebrum, suggesting regulation at the translational or post-translational levels. This inference is consistent with a recent report in L6 cells demonstrating that the induction of Glut3 protein in response to inhibition of oxidative phosphorylation by dinitrophenol occurs in the absence of any change in the content of Glut3 mRNA, and is mediated by increased stability of Glut3 protein w15x. Treatment with CoŽII. resulted in significant increases in the contents of both Glut1 and Glut3 in retina. Glut3 is expressed at significantly lower levels in retina than is Glut1 w35x. Glut1, however, is expressed at very high levels in cells comprising the blood–retinal barrier w32x. In vascular endothelial cells, Glut1 is present at a higher density in the ablumenal as compared to the lumenal
31
plasma membrane w19x. Because of Glut1’s localization and function, it has been proposed that alterations in the expression of this isoform may play a role in the pathogenesis of retinal complications of diabetes w16,19x. Results of the present study demonstrate that the increase in the content of Glut1 in retina is associated with increased Glut1 expression in its microvasculature. This latter finding suggests that exposure of retina to hypoxia in the presence of elevated blood glucose concentrations Žsuch as in diabetes. may well lead to large increases in the amount of glucose transported into this tissue. Previous reports indicate that the increase in Glut1 expression in cerebral microvessels in rats exposed to hypobaric hypoxia is associated with increased cerebral capillary density w10–12x. The increase in microvascularity is near-maximal at 1 week of exposure w11x and primarily reflects an increase in the length, rather than in the number, of capillaries w23x. The available evidence also indicates that cobalt chloride, acting in the presence of oxygen and without inhibiting oxidative phosphorylation, acts as a ‘surrogate’ of hypoxia and leads to enhanced expression of a set of hypoxia-responsive-genes, including the gene encoding VEGF and Glut1 w3,8,37x; this effect is mediated by activation of HIF-1, a basic-helix-loop-helix-PAS transcription factor w36,37x. Considerable evidence indicates that VEGF plays a critical role in the development of neovascularization w26,39x, but there is conflicting data as to whether VEGF alone is sufficient to produce new vessel growth. Both patients w1x and animals w25x with early diabetic retinopathy or other retinal diseases have elevated levels of retinal VEGF, without any evidence of neovascularization. Results of the present study indicate that although treatment with CoŽII. for 10–12 days increased the hematocrit, and stimulated the expression of Glut1 in cerebrum and retina and VEGF in cerebrum, the treatment did not change microvascular density in either tissue. This finding suggests that the presumed CoŽII.-induced activation of HIF-1, and the resulting increase in VEGF expression, while necessary, may not be sufficient to induce hypervascularity in the tissues examined. Whether or not the concentration of VEGF achieved a level sufficient to increase vascular density remains unclear. Alternatively, it is possible that unknown tissue factors that are released or activated secondary to inhibition of oxidative phosphorylation by hypoxia play an important role in increasing the microvascular density. Further studies are necessary to clarify the roles of inhibition of oxidative phosphorylation and stimulation of hypoxia-signaling pathway in the overall adaptive response to hypoxia.
Acknowledgements We are grateful to the late Dr. Ora M. Rosen of the Memorial Sloan-Kettering Cancer Center for supplying full-length rat GLUT1 cDNA. Human GLUT3 cDNA was
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kindly provided by Dr. Graeme I. Bell of the Howard Hughes Medical Institute at the University of Chicago. This study was supported in part by grants from the Diabetes Association of Greater Cleveland, and by National Institutes of Health aDK45945 to F. Ismail-Beigi, and aEY003000 to T.S. Kern. Dr. Badr was supported by a grant from the Government of Egypt and Dr. J. Tang is a recipient of a fellowship from Lions Club International Sightfirst program administered by the American Diabetes Association.
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w20x w21x
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