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Down-regulation of glucose transport by elevated extracellular glucose concentrations in cultured rat aortic smooth muscle cells does not normalize intracellular glucose concentrations
Down-regulation of glucose transport by elevated extracellular glucose concentrations in cultured rat aortic smooth muscle cells does not normalize intracellular glucose concentrations RANDY L. H O W A R D INDIANAPOLIS, INDIANA
Vascular disease is a prominent complication of diabetes mellitus, and hyperglycemia has been implicated as a risk factor for the development of these vascular complications. It has previously been suggested that down-regulation of glucose transport in response to hyperglycemia might serve a protective role by decreasing intracellular glucose concentrations. In the present study, regulation of glucose transport by extracellular glucose concentrations was investigated in cultured rat vascular smooth muscle cells (VSMCs). Confluent quiescent VSMCs were exposed to medium containing either normal (5 mmol/L) or elevated (20 mmol/L) extracellular glucose concentrations for 24 hours. VSMCs exposed to elevated extracellular glucose concentrations (with or without serum) for 24 hours exhibited significant decreases in 2-deoxyglucose (2-DG) and D-glucose uptake rates. This decreased glucose transport was associated with a decrease in the Vr, ox of D-glucose transport without a change in KM. In the absence of serum, a decrease in the quantity of GLUT-I transport protein at the plasma membrane was noted in cells exposed to elevated extracellular glucose concentrations for 24 hours. Intracellular glucose concentrations were estimated by using two methods, and the results revealed significantly higher intracellular glucose concentrations in the cells exposed to elevated extracellular glucose concentrations for 24 hours. These results suggest that down-regulation of glucose transport in cultured VSMCs exposed to elevated extraceilular glucose concentrations for 24 hours does not occur to an extent that normalizes intracellular glucose concentrations. This prolonged increase in intracellular glucose concentrations and the potential associated toxicity may explain the increased incidence of vascular complications in patients with diabetes mellitus. (J LABCklN MED 1996;127:504-
15) Abbreviations: BSA = bovine serum albumin; 2-DG = 2-deoxyglucose; ITS/AA = insulintransferrin-selenium-ascorbic acid; KRH = Krebs-Ringer-HEPES(N-2-hydroxyethylpiperazine-N-2ethanesulfonic acid) buffer; MEM = minimal essential medium; PBS = phosphate-buffered saline solution; VSMC = vascular smooth muscle cell
From the Department of Medicine, NephrologyDivision, Indiana University School of Medicine. Supported by Physician Scientist Award DK 02116 from the National Institutes of Health. Submitted for publication Aug. 23, 1995;revision submitted Dec. 13, 1995; accepted Dec. 19, 1995. Reprint requests: Randy L. Howard, MD, Assistant Professor of Medicine, Nephrology Section, Indiana University School of Medicine, Wishard Memorial Hospital, 1001 W. 10th St., OPW526, Indianapolis, IN 46202-2879. Copyright © 1996 by Mosby-YearBook, Inc. 0022-2143/96 $5.00 + 0 5/1/72641 504
iabetes mellitus is a multisystem disease in which the vascular system is prominently affected, l s Small and large vessel disease is the major cause of mortality and morbidity in patients with diabetes mellitus. Patients with diabetes mellitus have an increased prevalence of cardiovascular disease (coronary heart disease, cerebrovascular accidents, and hypertension), peripheral vascular disease, and rnicrovascular disease (retinopathy and nephropathy). Hyperglycemia has been implicated as a risk factor for the development of these vascular
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complications. 1'9-11 The cellular mechanisms and alterations in metabolism that may be associated with hyperglycemia remain to be defined. Possible mechanisms implicating hyperglycemia in the development of vascular disease include (1) polyol pathway activation and related imbalances in myoinositol metabolism, (2) accumulation of amphipathic lipids and associated changes in enzyme activities, (3) nonenzymatic glycation, and (4) reduced intracellular redox state resulting from a combination of the above events] A key event in the control and regulation of intracellular glucose and subsequent metabolites is the rate at which glucose enters cells. This glucose uptake process occurs by carrier-mediated facilitated diffusion across the plasma membrane, with subsequent phosphorylation of glucose by glucokinase or hexokinase to maintain the diffusion gradient. A family of glucose transport glycoproteins exists, 12 and cultured VSMCs express only the GLUT-1 glucose transport protein. 13'14 The rate of glucose uptake and expression of GLUT-1 protein can be regulated acutely and chronically by a variety of factors including serum, growth factors, transformation, steroids and nutrients. ~5-21 Kaiser et al. 14 have reported that chronic hyperglycemia down-regulates glucose transport and the glucose transporter in cultured bovine and human arterial smooth muscle cells. They suggest that this down-regulation of glucose transport might serve as a protective mechanism against possible adverse effects of increased intracellular glucose. Down-regulation of glucose transport in the setting of hyperglycemia would only serve a protective role if intracellular glucose concentrations were significantly decreased or normalized. Previous studies have not investigated the effect of glucose transport down-regulation on intracellular glucose concentrations. In the present study we have evaluated the regulation of glucose transport by extracellular glucose concentrations in cultured rat aortic smooth muscle cells. The results indicate that cultured VSMCs exposed to elevated extracellular glucose concentrations for 24 hours--when compared with cells exposed to normal extracellular glucose concentrations-down-regulate glucose transport by decreasing the number of plasma membrane glucose transporters (GLUT-l). Intracellular glucose concentrations in the cells exposed to elevated extracellular glucose concentrations for 24 hours remained significantly elevated when compared with cells exposed to normal extracellular glucose concentrations. These results indicate that down-regulation of glucose trans-
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port in cultured VSMCs exposed to 24 hours of elevated extracellular glucose concentrations does not normalize the intracellular glucose concentration, and therefore these cells remain susceptible to the adverse effects of increased intracellular glucose concentration. METHODS Cell culture, Rat aortic smooth muscle cells were isolated by using a modified method originally described by Chamley et al.22 and previously used by our group.23 The thoracic aortas from 6 to 8 male Sprague-Dawley rats (225 to 250 gm) were incubated at 37° C for 30 minutes in 6.0 ml of Eagle MEM containing 2 mg/ml collagenase. After the adventitia was dissected, the aortas were minced with a sterile razor blade and incubated again at 37° C in Eagle MEM containing 2 mg/ml collagenase for 2.5 to 3 hours with continuous stirring. The resulting single-cell suspension was centrifuged for 5 minutes at 800 g, and the cell pellet was resnspended in fresh incubation medium without collagenase. This washing procedure was repeated twice. The cells were plated onto 75 cm2 culture flasks and grown at 37° C in a humidified atmosphere of 95% air and 5% CO 2. Eagle MEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 ixg/ml streptomycin, and 0.25 i~g/ml amphotericin was used as the culture medium. Cell viability Was checked by the exclusion of 0.3% trypan blue dye, and it always exceeded 95%. The cultures reached confluence after 7 to 10 days and were subcultured with trypsin-ethylenediaminetetraacetic acid (0.25% to 1.0%) treatment. Cells were positively identified as smooth muscle by indirect immunofluorescent staining for smooth muscle cell alpha actin with a mouse monoclonal antibody and an anti-mouse immunoglobulin G fluorescein isothiocyanate conjugate. Cells between passages 3 and 10 were used for all experiments. For experiments, cells were incubated with serum-free medium containing 5 ixg/ml insulin, 5 ixg/ml transferrin, 5 ng/ml selenium, and 1 mmol/L ascorbic acid (ITS/AA) for 48 hours to induce quiescence. Arterial smooth muscle cells in vivo and from freshly isolated vessels exhibit a high rate of aerobic glycolysis,24'2s and initial experiments revealed that confluent VSMCs consumed glucose from the medium rapidly, resulting in glucose depletion (data not shown). To prevent glucose depletion, 3.0 ml of fresh serum-free medium was added to the cells at 24, 32, and 40 hours (Fig. 1). With this model the medium glucose concentration varied between approximately 5 and 3 mmol/L during the final 24 hours of the 48-hour serumfree incubation. After 48 hours in the serum-free medium, fresh serum-free (protocol 1, Fig. 1) or serum-containing (protocol 2, Fig. 1) medium was added as noted for each experiment, and again the medium was changed every 8 hours to prevent glucose depletion. Medium biochemical determinations. Glucose concentrations in tissue culture medium were measured by using a glucose analyzer (model 2300 STAT; YSI Inc., Yellow Springs, Ohio). The detection range for glucose is 0 to
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Feed with 3.0 mls 5 mM ITS/AA --~ medium
Feed with 3.0 mls 5 mM ITS/AA mediumevery 8 hours for 24 hours
Protocol 1 Feed with 3.0 mls 5 or 20 mM ITS/AA medium every 8 hours for 24 hours
Protocol 2 Feed with 3.0 mls __~ 5 o r 2 0 m M 1 0 % FCS medium every 8 hours for 24 hours
Fig. 1. Schematic representation of cell culture methods used in the described experiments to prevent medium glucose depletion. ITS/AA-insulin, transferrin, selenium, and ascorbic acid were at concentrations noted in Methods. FCS, Fetal calf serum.
29.2 mmol/L, with a resolution of 0.1 mmol/L, pH, PCO2, PO2, and HCO3 in tissue culture medium were determined by using a blood gas analyzer (model BG-3; Instrumentation Laboratories, Lexington, Mass.). Medium samples were quickly aspirated from the tissue culture plates and transferred to precooled microcentrifuge tubes that were stored on ice until determinations were performed. Uptake of 2-DG. 2-DG uptake, which measures both glucose transport and phosphorylation, was used as the initial method to examine the glucose transport system in cultured VSMCs. For these uptake experiments, VSMCs were grown in 35 mm dishes. At the indicated time, the medium was aspirated from the cells, and they were washed once with 1 ml of KRH (121 mmol/L NaC1, 4.9 mmol/L KC1, 1.2 mmol/L MgSO4, 0.33 mmol/L CaC12, 12 mmol/L HEPES, pH 7.4) at 37 ° C. A 1 ml sample of uptake solution (KRH with 0.1 mmol/L 2-DG and 1 ixCi/ml tritiated 2-DG) was added to each dish for 3 minutes (uptake was linear for 5 minutes, data not shown) at 37 ° C. The cells were subsequently washed three times with i ml of ice-cold KRH buffer and solubilized with i ml of 1N NaOH. A 500 pA sample of this solution was used for beta scintillation counting, and the remaining 500 txl was neutralized with HC1 and used for protein determination. Five ~mol/L cytochalasin B was included in some uptake experiments to determine nonspecific diffusion, which was subtracted from total cellular uptake. 2-DG uptake measures the combination of transport by the glucose transport protein and phosphorylation by hexokinase. Initial D-glucose transport rate. Initial glucose transport rates were determined by using a modification of the method of Whitesell et al. 26 This method as performed measures only glucose transport and not phosphorylation. At the indicated time, the medium was aspirated and the cells were washed twice with 1 ml 37 ° C KRH containing 0.1 mmol/L glucose and 0.5% BSA. The cells were then incubated at 37 ° C in 600 ixl KRH containing 0.1 mmol/L glucose, 0.5 p~Ci/ml [methyl-14C] 3-O-methylglucose or 0.5 ~Ci/ml [U-~4C] D-glucose and 2 p~Ci/ml [1-3H] L-glucose. For determination of glucose-accessible cell water, cells
were incubated for 1 hour with 3-O-methylglucose, and for determination of initial glucose uptake rates, cells were incubated with D-glucose for 10 seconds. At the end of the incubation period, 2 ml of stop solution (ice-cold KRH containing 200 txmol/L phloretin) was added directly to the uptake solution and rapidly aspirated. Two additional 2 ml washes were performed, and then the cells were solubilized in 1 ml of 0.1N NaOH with shaking for 30 minutes. A 750 Ixl aliquot was used for beta scintillation counting, and the remainder was saved for protein determination. Uptake of carbon 14-labeled glucose was calculated as follows. Raw 14C-labeled glucose space (txl/dish) equals total 14C on the dish after washing (cpm/dish) divided by the concentration of 14C in the uptake solution (cpm/ixl). Raw tritiated L-glucose space 0xl/dish) is calculated as total tritium on the dish after washing (cpm/dish) divided by the concentration of tritium in the uptake solution (cpm/txl). Raw tritiated L-glucose space is then subtracted from the raw 14C-labeled glucose space to yield nonnormalized specific space (~l/disfi). This value is normalized to glucose-accessible intracellular water measured as specific 14C-labeled 3-O-methylglucose space (~l/dish) after a 1-hour incubation. Finally, this value is normalized to protein content per dish, The final units of specific glucose uptake are/~1 of specific glucose space per ixl of glucoseaccessible cell water. Kinetic analysis of glucose transport. These experiments were performed in essentially the same fashion as the initial glucose transport rate experiments, except that the concentrations of glucose utilized were 1, 3, 6, and 10 mmol/L and the a4C-labeled D-glucose was increased to 2 p~Ci/ml to offset lower uptakes with the higher cold glucose concentrations. KM and Vma= were calculated by using a Lineweaver-Burk analysis of the transport data. A minimum of three experiments with an N of 6 at each glucose concentration were performed to calculate the K M and Vm=. Calculation of intracellular glucose concentration. Intracellular glucose concentrations were determined by using a modification of the method of Foley et a l Y At the
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indicated time, the medium was aspirated and the cells were washed twice with 1 ml 37° C KRH containing 0.5% BSA. The cells were then incubated at 37° C for 30 minutes in 600 ixl KRH containing various concentrations of glucose or equiosmotic sucrose, 0.5 ixCi/ml 14C-labeled 3-O-methylglucose, and 2 ixCi/ml tritiated L-glucose. At the end of the 30-minute incubation, 2 ml of stop solution (ice-cold KRH containing 200 ixmol/L phloretin) was added directly to the uptake solution and rapidly aspirated. Two additional 2 ml washes were performed, and then the cells were solubilized in 1 ml of 0.1N NaOH with shaking for 30 minutes. A 750 ~1 aliquot was used for beta scintillation counting, and the remainder was saved for protein determination. These uptake data were used to calculate a ratio of steady-state intracellular 3-O-methylglucose uptake in the presence of glucose to that in the presence of equiosmotic sucrose. With this ratio and the K m for glucose transport, the intracellular glucose concentration (@) can be calculated from the following formula: Ratio = (1 + [Gi]/Km)/ (1 + [Go]/Km), where Gi = intracellular glucose concentration and G o = extracellular glucose concentration. Determination of intracellular glucose concentration with 2-3H-glucose.VSMCs were incubated with tracer
quantities of 2-3H-glucose during the final 8 hours of the 24-hour incubation with 5 or 20 mmol/L glucose. At the indicated time, the medium was aspirated and the cells washed 3 times with 1 ml of ice-cold KRH containing 200 ixmol/L phloretin. The cells were scraped into 1 ml of ice-cold 10 mmol/L Tris-C1 containing 0.5% Triton X-100 and homogenized with a syringe. Tritiated water and unreacted 2-3H-glucose were separated by using a modification of the method described by Hammerstedt. 2s Columns containing Dowex-l-borate were prepared and washed with water. A 500 ixl sample of the homogenized cell sample was applied to the column. Tritiated water was eluted with three 1 ml aliquots of water, and 2-3H-glucose was eluted with three i ml aliquots of 1N HC1. Preliminary experiments revealed that greater than 99% of the tritiated water and less than 1% of the tritiated glucose was recovered in the water elution, and less than 1% of the tritiated water and greater than 99% of the tritiated glucose was recovered in the HCI elution (data not shown). Biotinylation of plasma membranes. Biotinylation of plasma membranes was performed as a modification of the method described by Shetty et al. a9 At the indicated time, the cells were washed twice with 8 ml of ice-cold PBS, and then 1.5 ml of cold biotinylation buffer (120 mmol/L NaC1, 30 mmol/L NaHCO3, 5 mmol/L KC1, pH 8.5) containing 0.1 mg/ml of freshly added NHS-LC-biotin (Pierce, Rockford, Ill.) was added to each dish. The dishes were incubated at 4°C for 30 minutes with occasional swirling. The biotinylation buffer was then aspirated and the dishes were washed three times with 5 ml of buffer containing 140 mmol/L NaC1, 20 mmol/L Tris, and 5 mmol/L KC1 at pH 7.5. Cells from three dishes were then scraped and pooled in 1 ml of hypotonic homogenization buffer containing 10 mmol/L NaHCO 3 and 0.1 mmol/L
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Pefabloc SC (Boehringer Mannheim Corp., Indianapolis, Ind). After 10 minutes on ice, the cells were homogenized with 40 strokes in a glass-Teflon homogenizer. One milliliter of sample was transferred to a microcentrifuge tube, and 100 ~1 of buffer containing 1.5 mol/L NaC1 and 100 mmol/L Tris, pH 7.0, was added. The homogenate was spun for 15 seconds at maximum speed in an Eppendorf Microfuge (Brinkman Instruments, Westbury, N.Y.) to sediment nuclei. The resulting postnuclear supernatant was transferred to a microcentrifuge tube containing 50 ~1 of streptavidin agarose beads (Pierce) that had been sedimented after pre-equilibration with 1 ml of homogenization buffer. After gentle mixing by repeated inversion at 4° C for 30 minutes, the beads were pelleted at 3000 rpm in a refrigerated microcentrifuge at 4°C (International Equipment Co., Needham Heights, Mass.) and washed three times with 1 ml of homogenization buffer containing freshly added Pefabloc SC. The final pellets were resuspended in 120 ixl of ×1.2 Laemmli buffer lacking both mercaptoethanol and bromphenol blue and were incubated at 65 ° C for 30 minutes. The beads were once again pelleted, and the supernatant containing solubilized plasma membrane was removed and frozen. Protein content was determined the next day by using a BCA protein assay kit with BSA and appropriate concentrations of sodium dodecyl sulfate in the standards. Western blot analysis. Cell fractions were denatured by heating for 30 minutes at 60°C in the presence of Laemmli buffer containing 10 mmol/L dithiothreitol. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli, 3° with an 8% acrylamide running gel and a 4% acrylamide stacking gel. Proteins were transferred to nitrocellulose paper (0.2 ~m pore size; Schleicher and Schuell Co., Keene, N.H.) by using a Royal Genie electroblotter (Idea Scientific Co., Minneapolis, Minn.) with a 25 mmol/L Tris, 192 mmol/L glycine, 20% methanol buffer. The nitrocellulose was stained with napthol blue black to assess transfer of proteins. Filters were then blocked overnight with PBS containing 5 % BSA and 0.1% Tween 20. After blocking, filters were washed three times with PBS containing 0.1% Tween 20 and were incubated with a 1:2000 dilution of specific GLUT-1 antibody (East Acres Biologicals, Southbridge, Mass.) in PBS containing 3% BSA and 0.1% Tween 20 for 2 hours. The filter was again washed and the primary antibody detected by incubation with iodine 125-labeled protein A in PBS containing 0.1% Tween 20 for 2 hours. The filters were then washed, dried, and exposed to Kodak XAR-5 film. Band intensities were determined from autoradiograms by using a Seprascan 2001 (Integrated Separations Systems, Hyde Park, Mass.). Estimation of protein content. Total protein content was determined by using the bicinchoninic acid method (BCA Protein Assay Reagent; Pierce) with BSA as the standard. Statistics. Results are reported as the mean + SEM. N equals the number of wells assayed. Statistical analysis was performed by using unpaired Student's t test or analysis of
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Protocol 1
Fig. 2. Effect of chronic hyperglycemia on 2-DG uptake in cultured VSMCs. Cells were treated as outlined in Fig. 1, and 2-DG uptake was performed as described in Methods. Open column, 5 mmol/L glucose; hatched column, 20 mmol/L glucose; shadedcolumn, 5 mmol/L glucose plus 15 mmol/L mannitol. N = 6 for each group. *p < 0.01 versus 5 mmol/L glucose group of the same protocol.
Table I. Media parameters obtained by using the two described protocols
Protocol 1 2
Starting glucose concentration Low High Low High
Medium glucose at completion of experiment (mmol/L) 3.5 17.9 1.4 15.2
± 0.1 ± 0,1 ± 0.1 _+ 0.1
pH 7.22 7.23 7.07 7.06
± ± ± +
0.001 0.001 0.005 0.009
Pco2 (torr) 41.8 39.7 51.2 50.5
± 1.1 ± 0,8 ± 0.9 _+ 0.6
P2 (torr) 129 144 115 122
± ± ± +
6.8 5.7 2.7 1.6
HC03 (mmol/L)
17.3 ± 0.2 16.6 ± 0.1 14.8 ± 0.2 14.5 + 0.3
Cultured rat aortic smooth muscle cells were treated as described in Methods and in Fig. 1. N = 6 for all groups.
variance, as indicated in the Results section, andp values less than 0.05 were considered significant. To investigate whether there was a significant difference in the cytochalasin B-sensitive uptake of 2-DG in VSMCs, a two-way analysis of variance with interaction was computed. Specifically, if the interaction is significant, then there exists evidence of a difference in the cytochalasin B-sensitive uptake of 2-DG between the various extraceIlular glucose concentration groups. RESULTS Cell culture methods. Previous investigations have noted that freshly isolated porcine arteries exhibit high rates of aerobic glycolysis.24'25 Cultured rat aortic smooth muscle cells exhibited similar characteristics, resulting in rapid medium glucose utilization and lactate production (data not shown). To avoid medium glucose depletion, which up-regulates the glucose transport system, the cell culture and feeding protocol outlined in Fig. 1 was utilized for all experiments. Medium was changed every 8 hours
to prevent medium glucose depletion and to maintain a more constant pH. As shown in Table I, the most physiologic medium conditions were obtained when cells were cultured in the serum-free (ITS/ AA) medium. When serum was added to the medium, glucose utilization increased and subsequent lactate production increased, resulting in lower medium glucose and pH values. Uptake of 2-DG. As an initial method to examine the chronic effect of elevated extracellular glucose concentrations on the glucose transport system, we utilized the 2-DG uptake assay, which measures both glucose transport and phosphorylation. As shown in Fig. 2, 2-DG uptake was significantly decreased (two-way analysis of variance with interaction) in VSMCs exposed to elevated extracellular glucose concentrations for 24 hours. The percent decrease in 2-DG uptake was similar in the presence or absence of serum (approximately a 36% decrease), and it can be seen that the rate of 2-DG uptake was higher in the cells exposed to serum.
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Fig. 3. Effect of chronic hyperglycemia on initial D-glucose uptake rate in cultured VSMCs. Cells were treated as outlined in Fig. 1, and D-glucose uptake was performed as described in Methods. A, Protocol 1. B, Protocol 2. Open column, 5 mmol/L glucose; hatched column, 20 mmol/L glucose; shaded column, 5 mmol/L glucose plus 15 mmol/L mannitol. N = 6 for each group in each experiment. *p < 0.01 versus 5 mmol/L glucose group of the same experiment.
These results suggest that either a decrease in glucose transport or a decrease in glucose phosphorylation occurs in the VSMCs exposed chronically to hyperglycemic medium. Initial D-glucose transport rate. To investigate whether the alterations in 2-DG uptake were the result of changes in glucose transport or phosphorylation, we utilized the initial D-glucose transport assay. By utilizing the natural substrate for the glucose transporter (D-glucose) and short uptake periods (10 seconds), we were able to directly estimate the glucose transport parameters. As shown in Fig.
3, the initial D-glucose transport rate was significantly decreased (Student's t test) in VSMCs exposed to hyperglycemic medium for 24 hours. Again, it can be seen that glucose transport was higher in the cells exposed to serum and that the decrease in glucose transport occurred in the presence or absence of serum. These results suggest that a decrease in glucose transport occurs in VSMCs exposed chronically to hyperglycemic medium. Mechanism of decreased glucose transport. We next set out to determine the mechanism responsible for the decrease in glucose transport in VSMCs exposed
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GLUT - 1
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l
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Fig. 4. Effect of chronic hyperglycemia on GLUT-1 transport protein in plasma membrane fraction. Cells were treated as outlined in protocol 1 of Fig. 1, and plasma membrane proteins were biotinylated, immunoprecipitated, and immunoblotted for the GLUT-1 protein as described in Methods. A 10 ~g sample of plasma membrane protein from three pooled 100 mm plates was run in each lane. The four lanes in each group represent separate experiments on different passages of cells. Molecular weight markers are indicated on the right side of the figure. The numbers below the autoradiograph represent the arbitrary densitometry units for each group (p value < 0.05).
Table II. Kinetic analysis of glucose transport in culfured VSMCs Protocol 1 2
Glucose concentration 5 20 5 20
mmol/L mmol/L mmol/L mmol/L
Km (retool/L) 1.74 1.95 2.86 2.77
± 0.12 --- 0.14 ± 0.08 --- 0.10
Vma x
p value NS NS
(pmol/sec/mg protein) 101 53 426 222
± 2.0 _+ 1.7 ± 8.7 -+ 6.4
p value <0.01 <0.01
Cultured rat aortic smooth muscle ceils were treated as described in Methods and in Fig, 1, Kinetic analysis was performed as described in Methods. The results are pooled from three separate passages of ceils with N = 6 for each group in each passage.
chronically to hyperglycemic medium. As shown in Table II, kinetic analysis of glucose transport revealed a significant decrease in the Vma x for glucose transport without a significant change in the K m for glucose transport in VSMCs exposed to hyperglycemic medium for 24 hours. The kinetic changes in glucose transport were similar in the presence or absence of serum, and the Km and Vmax for glucose transport were higher in the VSMCs exposed to serum for 24 hours. This decrease in Vmax could be the result of either a decrease in the absolute number of glucose transporters or a decrease in the transport rate of the glucose transporters present at the plasma membrane. To examine this question, we used biotinylation of surface membrane proteins followed by immunoprecipitation of the labeled proteins and immunoblotting of the GLUT-1 transport protein as described by Shetty et al.29 The results of these experiments are shown in Fig. 4. The figure shows that the quantity of GLUT-1 transport protein present at the plasma membrane is decreased in
VSMCs exposed to hyperglycemic medium for 24 hours. Densitometry measurements were performed on the blot in Fig. 4; the results are shown below the figure, and they confirm a decrease in plasma membrane GLUT-1 transport protein in VSMCs exposed chronically to hyperglycemic medium. Effect of alterations in glucose transport on intracellulor
glucose concentrations. The final issue that we wanted to examine concerned the effect of decreased glucose transport in VSMCs exposed chronically to hyperglycemic medium on intracellular glucose concentrations. To examine this question, we used two methods to estimate intracellular glucose concentrations. The first method utilized steady-state countertransport kinetics to measure the intracellular glucose concentration indirectly.27 As shown in Fig. 5, A, the ratio of steady-state intracellular 14C-labeled 3-O-methylglucose in the presence of glucose to that in the presence of equiosmotic sucrose is lower at both buffer glucose concentrations used in VSMCs exposed to hyperglycemic medium in the absence of
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*# Calculated lntracellular Glucose Concentration (mM)
0 Medium Glucose Concentration (mM) Buffer Glucose Concentration (mM)
5 5
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Fig. 5. Effect of chronic hyperglycemia on indirectly determined intracellular glucose concentration. Cells were treated as outlined in protocol 1 of Fig. 1, and steady-state intracellular 3-O-methylglucose concentrations were determined as described in Methods. A, Ratio of intracellular 3-O-methylglucose in the presence of the indicated buffer glucose concentrations or equiosmotic sucrose. B, Intracellular glucose concentration calculated from the ratios obtained in A and the I ~ values from Table II. The results were obtained from three experiments with N = 3 for each experiment. *p < 0.01 versus same medium glucose concentration and 5 mmol/L buffer glucose concentration. #p < 0.01 versus 5 mmol/L medium glucose concentration and 5 mmol/L buffer glucose concentration.
s e r u m for 24 hours. T h e lower ratio in the V S M C s e x p o s e d to chronically e l e v a t e d extracellular glucose c o n c e n t r a t i o n s (at e i t h e r buffer glucose c o n c e n t r a tion) is consistent with the d e c r e a s e d glucose transp o r t n o t e d in e a r l i e r e x p e r i m e n t s . T h e c o r r e s p o n d -
ing c a l c u l a t e d i n t r a c e l l u l a r glucose c o n c e n t r a t i o n s a r e shown in Fig. 5, B, a n d r e v e a l that in the face of d e c r e a s e d glucose t r a n s p o r t in V S M C s e x p o s e d chronically to h y p e r g l y c e m i c m e d i u m , the intracellular glucose c o n c e n t r a t i o n r e m a i n s h i g h e r t h a n in
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Fig. 6. Effect of chronic hyperglycemia on indirectly determined intracellular glucose concentration. Cells were treated as outlined in protocoI 2 of Fig. 1, and steady-state intracellular 3-O-methylglucose concentrations were determined as described in Methods. A, Ratio of intracellular 3-O-methylgtucose in the presence of the indicated buffer glucose concentrations or equiosmotic sucrose. B, Intracellular glucose concentration calculated from the ratios obtained in A and the K~ values from Table II. The results were obtained from three experiments with N = 3 for each experiment. *p < 0.01 versus same medium glucose concentration and 5 mmol/L buffer glucose concentration. # p < 0.01 versus 5 mmol/L medium glucose concentration and 5 mmol/L buffer glucose concentration.
cells exposed to normoglycemic medium (compare the bars labeled 5/5 and 20/20, which represent the actual conditions to which the cells were exposed). Fig. 6 shows that similar results were seen in cells incubated in the presence of serum, although the degree of down-regulation of intracellular glucose concentration appears to be less dramatic. When Figs. 5 and 6 are compared, it can be noted that intracellular glucose concentrations are significantly higher in cells incubated in the presence of serum. A second method for estimating intracellular glucose was also used. This method utilizes tritiated
glucose as a tracer, and it requires separation of the tritiated glucose from tritiated water, generated in the metabolism of glucose-6-phosphate to fructose6-phosphate, by using Dowex chromatography. As shown in Fig. 7, VSMCs exposed to hyperglycemic medium for 24 hours exhibited significantly higher (Student's t test) intracellular glucose concentrations than cells exposed to normoglycemic medium. As noted for the first method, intracellular glucose concentrations were higher in cells exposed to serum, and the higher intracellular glucose concentrations seen with hyperglycemic culture conditions oc-
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Protocol 1
Protocol 2
Fig. 7. Effect of chronic hyperglycemia on intracellular glucose concentration determined by using tritiated glucose. Cells were treated as outlined in Fig. 1, and intracellular tritiated glucose concentrations were determined as described in Methods. Open column, 5 mmol/L glucose; hatched column, 20 mmol/L glucose. N = 6 for each group in each experiment. *p < 0.01 versus 5 mmol/L glucose group of the same protocol.
curred in the presence or absence of serum. The results from these two sets of studies suggest that intracellular glucose concentrations remain higher in VSMCs exposed chronically to hyperglycemic medium in spite of decreased glucose transport. DISCUSSION
In the present study we have shown that chronic elevation of extracellular glucose concentration (20 mmol/L glucose for 24 hours) leads to down-regulation of glucose transport by decreasing the number of GLUT-1 glucose transporters present at the plasma membrane. In addition , we have utilized two separate methods to show that this down-regulation of glucose transport does not result in normalization of intracellular glucose concentration after a 24hour exposure to elevated extracellular glucose concentrations. These results are similar to those reported by Kaiser et al} 4 but provide additional important information regarding the regulation of glucose transport in cultured VSMCs. Kaiser et al. reported a decrease in glucose transport and crude membrane GLUT-1 protein in bovine and human smooth muscle cells exposed to hyperglycemic medium for 24 hours. Cells exposed to the two lowest medium glucose concentrations (1.2 and 5.5 mmol/L) in this study may have been exposed to depleted or neardepleted medium glucose conditions at the time the assays of glucose transport were performed because of the culture protocol used. If this situation occurred, resulting in up-regulation of glucose trans-
port caused by medium glucose depletion, the results would be difficult to interpret. In contrast, we have utilized a culture protocol designed specifically to prevent medium glucose depletion and to provide variation in medium glucose concentrations and pH over a narrow range (see Fig. 1 and Table I). Using the described cell culture protocol, we have also found that chronic elevation of extracellular glucose concentrations results in down-regulation of glucose transport as measured by both 2-DG uptake (Fig. 2) and initial D-glucose transport rates (Fig. 3). While 2-DG uptake is technically easier because of the prolonged uptake periods, it measures both transport and phosphorylation of the non-metabolizable 2-DG. Initial D-glucose transport reflects only the transport of glucose when short time points are used (10 seconds in this study). It should be noted that the down-regulation of glucose transport was much more dramatic in Kaiser's studies, and again this may reflect the culture protocol used. Similar to Kaiser et al., 14 we have also found that the decrease in glucose transport is the result of a decrease in Vm~x for glucose transport (Table II). Of note, our calculated K~ values for glucose transport are significantly higher than those reported by Kaiser et al. (1.75 to 1.95 mmol/L versus approximately 0.85 mmol/L). We believe these differences reflect the different methods used. Kaiser et al. performed kinetic analysis of glucose transport by using 2-DG uptake, whereas we utilized initial D-glucose uptake rates. We chose to use D-glucose because it is the natural substrate for the glucose transporter and
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therefore does not perturb intracellular metabolism. In contrast, 2-DG can be transported and phosphorylated but not metabolized further. Using a recently developed technique to label surface membrane proteins with biotin followed by immunoprecipitation and immunoblotting for the GLUT-1 protein, we have been able to show that the decreased Vm~x of glucose transport is associated with a decrease in the quantity of GLUT-1 glucose transporter at the plasma membrane (Fig. 4). Kaiser et al. were unable to find a difference in the quantity of GLUT-1 transport protein in a crude membrane fraction when comparing cells exposed to 5.5 and 22 mmol/L glucose. Kaiser et al. 14 suggest that "the ability of VSMCs to down-regulate glucose transport in response to chronic hyperglycemia may serve as a protective mechanism against possible adverse effects of increased intracellular glucose." We are not aware of any measurements or estimates of intracellular glucose in VSMCs that actually address this issue. We have used two methods to indirectly (steady-state intracellular 3-O-methylglucose concentrations) and directly (tritiated glucose) estimate intracellular glucose concentrations. Although the two methods yielded different results regarding the magnitude of the differences, both methods revealed that intracellular glucose concentrations were significantly elevated in VSMCs exposed to chronic elevations of extracellular glucose concentrations when compared with cells exposed to normal extracellular glucose concentrations (Figs. 5 through 7). Although the intracellular concentration of glucose that is "toxic" to the cell has not been documented, and although we have no measurements of potentially toxic glucose metabolites (sorbitol, myoinositol, amphipathic lipids, protein kinase C) to document toxicity in our system, we believe that the potential for toxicity related to increased intracellular glucose concentrations remains even after 24 hours of elevated extracellular glucose concentrations. This inability of VSMCs to normalize intracellular glucose concentrations, resulting in prolonged increases in intracellular glucose concentrations, may explain why the vascular system is so prominently affected in patients with diabetes mellitus and hyperglycemia. Another interpretation of these data would be that the elevated intracellular glucose concentrations result from decreased metabolism of glucose to toxic metabolites. This response would protect the cell from toxic glucose metabolites but raise the intracellular glucose concentration. Detailed measurements of glucose metabolites will be required to clarify this issue. The presence of measurable intracellular concen-
trations of free glucose in all experimental conditions used indicates that glucose metabolism and not glucose transport is the rate-limiting step for glucose uptake in cultured smooth muscle cells. Clarification of the changes in glucose metabolism associated with elevated extracellular glucose concentrations may provide insight into the vascular disease associated with diabetes mellitus and hyperglycemia. In summary, we have shown that exposure of cultured VSMCs to chronically elevated extracellular glucose concentrations results in down-regulation of glucose transport that is the result of a decreased quantity of GLUT-1 glucose transport protein at the plasma membrane. This down-regulation of glucose transport does not result in normalization of intraceUular glucose concentration after a 24-hour exposure to elevated extracellular glucose concentrations. Down-regulation of glucose transport does not appear to normalize intracellular glucose concentrations over the time period studied, and these results may explain the susceptibility of VSMCs to the toxic effects of hyperglycemia. I thank Daniel J, Homco, BS, for technical assistance. REFERENCES
1. Viberti GC, Messent J. Introduction: hypertension and diabetes. Critical combination for micro- and macrovascular disease. Diabetes Care 1991;14(suppl 4):4-7. 2. Jarrett RJ, Shipley MJ. Mortality and associated risk factors in diabetics. Acta Endocrinol 1985;110(suppl 272):21-6. 3. Jarret RJ, Keen H, Chakrokati A. Diabetes, hyperglycemia and arterial disease. In: Keen H, Jarret RJ, eds. Complications of diabetes. London: Edward Arnold, 1982:79-84. 4. Krolewski AS, Warren JH, Rand LI, Kahn CR. Epidemiological approach to the etiology of type 1 diabetes and its complications. N Engl J Med 1987;317:1390-8. 5. Pirart J. Diabetes mellitus and its degenerative complications: a prospective study of 4400 patients observed between 1947-1973. Diabetes Care 1978;1:168-88. 6. The DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977-86. 7. Pugliese G, Tilton RG, Williamson JR. Glucose-induced metabolic imbalances in the pathogenesis of diabetic vascular disease. Diabetes Metab Rev 1991;7:35-59. 8. Wilson PW, Cupples LA, Kannel WB. Is hyperglycemia associated with cardiovascular disease? The Framingham study. Am Heart J 199I;121:586-90. 9. Pell S, D'Aionzo CA. Some aspects of hypertension in diabetes mellitus. JAMA 1967;202:104-10. 10. Jarrett RJ, Keen H, McCartney M, et al. Glucose tolerance and blood pressure in two population samples: their relation to diabetes mellitus and hypertension. Int J Epidemiol 1978; 7:15-24. 11. Christlieb AR, Warram JH, Krolewski AS, et al. Hypertension: the major risk in juvenile-onset insulin-dependent diabetics. Diabetes 1981;30(suppl 2):90-6.
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12. Kahn BB. Facilitative glucose transporters: regulatory mechanisms and dysregulation in diabetes. J Clin Invest 1992;89: 1367-4. 13. Low BC, Ross IK, Grigor MR. Angiotensin II stimulates glucose transport activity in cultured vascular smooth muscle cells. J Biol Chem 1992;267:20740-5. 14. Kaiser N, Sasson S, Feener EP, et al. Differential regulation of glucose transport and transporters by glucose in vascular endothelial and smooth muscle cells. Diabetes 1993;42:80-9. 15. Hiraki Y, Rosen OM, Birnbaum MJ. Growth factors rapidly induce expression of the glucose transporter gene. J Biol Chem 1988;263:13655-62. 16. White MK, Weber MJ. Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms. Mol Cell Biol 1988;8:138-44. 17. Haspel HC, Wilk EW, Birnbaum MJ, Cushman SW, Rosen OM. Glucose deprivation and hexose transporter polypeptides of murine fibroblasts. J Biol Chem 1986;261:6778-89. 18. Haspel HC, Mynarcik DC, Ortiz PA, Honkanen RA, Rosenfeld MG. Glucose deprivation induces the selective accumulation of hexose transporter protein GLUT-1 in the plasma membrane of normal rat kidney cells. Mol Endocrinol 1991;5:61-72. 19. Kalckar HM, Ullrey DB. Further clues concerning the vectors essential to regulation of hexose transport, as studied in fibroblast cultures from a metabolic mutant. Proc Natl Acad Sci USA i984;81:1126-9. 20. Yamada K, Tillotson LG, Isselbacher KJ. Regulation of hexose carriers in chicken embryo fibroblasts. Effect of glucose starvation and role of protein synthesis. J Biol Chem 1983; 258:9786-92. 21. Kitzman HH, McMahon RJ, Williams MG, Frost SC. Effect
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of glucose deprivation on GLUT 1 expression in 3T3-L1 adipocytes. J Biol Chem 1993;268:1320-5. Chamley JH, Campbell GR, McConnel JD. Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and subculture. Cell Tissue Res 1977;177:503-22. Soleimani M, Howard RL. Presence of chloride-formate exchange in vascular smooth muscle and cardiac cells. Circ Res 1994;74:48-55. Lynch RM, Paul RJ. Compartmentation of glycolytic and glycogenolytic metabolism in vascular smooth muscle. Science 1983;222:1344-6. Paul RJ. Chemical energetics of vascular smooth muscle. In: Bohr DF, Somylo AP, Sparks H, eds. Handbook of physiology. Baltimore: Williams and Wilkins, 1981;201-35. Whitesell RR, Regen DM, Pelletier D, Abumrad NA. Evidence that downregulation of hexose transport limits intracellular glucose in 3T3-L1 fibroblasts. Diabetes 1990;39:122834. Foley JE, Cushman SW, Salans LB. Intracellular glucose concentration in small and large rat adipose cells. Am J Physiol 1980;238:E180-5. Hammerstedt RH. The use of Dowex-l-borate to separate 3HOH from 2-3H-glucose. Anal Biochem 1973;56:292-3. Shetty M, Loeb JN, Vikstrom K, Ismail-Beigi F. Rapid activation of GLUT-1 glucose transporter following inhibition of oxidative phosphorylation in clone 9 cells. J Biol Chem 1993; 268:17225-32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 1970;227: 680-5.
Vascular disease is a prominent complication of diabetes mellitus, and hyperglycemia has been implicated as a risk factor for the development of these vascular complications. It has previously been suggested that down-regulation of glucose transport in response to hyperglycemia might serve a protective role by decreasing intracellular glucose concentrations. In the present study, regulation of glucose transport by extracellular glucose concentrations was investigated in cultured rat vascular smooth muscle cells (VSMCs). Confluent quiescent VSMCs were exposed to medium containing either normal (5 mmol/L) or elevated (20 mmol/L) extracellular glucose concentrations for 24 hours. VSMCs exposed to elevated extracellular glucose concentrations (with or without serum) for 24 hours exhibited significant decreases in 2-deoxyglucose (2-DG) and D-glucose uptake rates. This decreased glucose transport was associated with a decrease in the Vr, ox of D-glucose transport without a change in KM. In the absence of serum, a decrease in the quantity of GLUT-I transport protein at the plasma membrane was noted in cells exposed to elevated extracellular glucose concentrations for 24 hours. Intracellular glucose concentrations were estimated by using two methods, and the results revealed significantly higher intracellular glucose concentrations in the cells exposed to elevated extracellular glucose concentrations for 24 hours. These results suggest that down-regulation of glucose transport in cultured VSMCs exposed to elevated extraceilular glucose concentrations for 24 hours does not occur to an extent that normalizes intracellular glucose concentrations. This prolonged increase in intracellular glucose concentrations and the potential associated toxicity may explain the increased incidence of vascular complications in patients with diabetes mellitus. (J LABCklN MED 1996;127:504-
15) Abbreviations: BSA = bovine serum albumin; 2-DG = 2-deoxyglucose; ITS/AA = insulintransferrin-selenium-ascorbic acid; KRH = Krebs-Ringer-HEPES(N-2-hydroxyethylpiperazine-N-2ethanesulfonic acid) buffer; MEM = minimal essential medium; PBS = phosphate-buffered saline solution; VSMC = vascular smooth muscle cell
From the Department of Medicine, NephrologyDivision, Indiana University School of Medicine. Supported by Physician Scientist Award DK 02116 from the National Institutes of Health. Submitted for publication Aug. 23, 1995;revision submitted Dec. 13, 1995; accepted Dec. 19, 1995. Reprint requests: Randy L. Howard, MD, Assistant Professor of Medicine, Nephrology Section, Indiana University School of Medicine, Wishard Memorial Hospital, 1001 W. 10th St., OPW526, Indianapolis, IN 46202-2879. Copyright © 1996 by Mosby-YearBook, Inc. 0022-2143/96 $5.00 + 0 5/1/72641 504
iabetes mellitus is a multisystem disease in which the vascular system is prominently affected, l s Small and large vessel disease is the major cause of mortality and morbidity in patients with diabetes mellitus. Patients with diabetes mellitus have an increased prevalence of cardiovascular disease (coronary heart disease, cerebrovascular accidents, and hypertension), peripheral vascular disease, and rnicrovascular disease (retinopathy and nephropathy). Hyperglycemia has been implicated as a risk factor for the development of these vascular
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complications. 1'9-11 The cellular mechanisms and alterations in metabolism that may be associated with hyperglycemia remain to be defined. Possible mechanisms implicating hyperglycemia in the development of vascular disease include (1) polyol pathway activation and related imbalances in myoinositol metabolism, (2) accumulation of amphipathic lipids and associated changes in enzyme activities, (3) nonenzymatic glycation, and (4) reduced intracellular redox state resulting from a combination of the above events] A key event in the control and regulation of intracellular glucose and subsequent metabolites is the rate at which glucose enters cells. This glucose uptake process occurs by carrier-mediated facilitated diffusion across the plasma membrane, with subsequent phosphorylation of glucose by glucokinase or hexokinase to maintain the diffusion gradient. A family of glucose transport glycoproteins exists, 12 and cultured VSMCs express only the GLUT-1 glucose transport protein. 13'14 The rate of glucose uptake and expression of GLUT-1 protein can be regulated acutely and chronically by a variety of factors including serum, growth factors, transformation, steroids and nutrients. ~5-21 Kaiser et al. 14 have reported that chronic hyperglycemia down-regulates glucose transport and the glucose transporter in cultured bovine and human arterial smooth muscle cells. They suggest that this down-regulation of glucose transport might serve as a protective mechanism against possible adverse effects of increased intracellular glucose. Down-regulation of glucose transport in the setting of hyperglycemia would only serve a protective role if intracellular glucose concentrations were significantly decreased or normalized. Previous studies have not investigated the effect of glucose transport down-regulation on intracellular glucose concentrations. In the present study we have evaluated the regulation of glucose transport by extracellular glucose concentrations in cultured rat aortic smooth muscle cells. The results indicate that cultured VSMCs exposed to elevated extracellular glucose concentrations for 24 hours--when compared with cells exposed to normal extracellular glucose concentrations-down-regulate glucose transport by decreasing the number of plasma membrane glucose transporters (GLUT-l). Intracellular glucose concentrations in the cells exposed to elevated extracellular glucose concentrations for 24 hours remained significantly elevated when compared with cells exposed to normal extracellular glucose concentrations. These results indicate that down-regulation of glucose trans-
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port in cultured VSMCs exposed to 24 hours of elevated extracellular glucose concentrations does not normalize the intracellular glucose concentration, and therefore these cells remain susceptible to the adverse effects of increased intracellular glucose concentration. METHODS Cell culture, Rat aortic smooth muscle cells were isolated by using a modified method originally described by Chamley et al.22 and previously used by our group.23 The thoracic aortas from 6 to 8 male Sprague-Dawley rats (225 to 250 gm) were incubated at 37° C for 30 minutes in 6.0 ml of Eagle MEM containing 2 mg/ml collagenase. After the adventitia was dissected, the aortas were minced with a sterile razor blade and incubated again at 37° C in Eagle MEM containing 2 mg/ml collagenase for 2.5 to 3 hours with continuous stirring. The resulting single-cell suspension was centrifuged for 5 minutes at 800 g, and the cell pellet was resnspended in fresh incubation medium without collagenase. This washing procedure was repeated twice. The cells were plated onto 75 cm2 culture flasks and grown at 37° C in a humidified atmosphere of 95% air and 5% CO 2. Eagle MEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 ixg/ml streptomycin, and 0.25 i~g/ml amphotericin was used as the culture medium. Cell viability Was checked by the exclusion of 0.3% trypan blue dye, and it always exceeded 95%. The cultures reached confluence after 7 to 10 days and were subcultured with trypsin-ethylenediaminetetraacetic acid (0.25% to 1.0%) treatment. Cells were positively identified as smooth muscle by indirect immunofluorescent staining for smooth muscle cell alpha actin with a mouse monoclonal antibody and an anti-mouse immunoglobulin G fluorescein isothiocyanate conjugate. Cells between passages 3 and 10 were used for all experiments. For experiments, cells were incubated with serum-free medium containing 5 ixg/ml insulin, 5 ixg/ml transferrin, 5 ng/ml selenium, and 1 mmol/L ascorbic acid (ITS/AA) for 48 hours to induce quiescence. Arterial smooth muscle cells in vivo and from freshly isolated vessels exhibit a high rate of aerobic glycolysis,24'2s and initial experiments revealed that confluent VSMCs consumed glucose from the medium rapidly, resulting in glucose depletion (data not shown). To prevent glucose depletion, 3.0 ml of fresh serum-free medium was added to the cells at 24, 32, and 40 hours (Fig. 1). With this model the medium glucose concentration varied between approximately 5 and 3 mmol/L during the final 24 hours of the 48-hour serumfree incubation. After 48 hours in the serum-free medium, fresh serum-free (protocol 1, Fig. 1) or serum-containing (protocol 2, Fig. 1) medium was added as noted for each experiment, and again the medium was changed every 8 hours to prevent glucose depletion. Medium biochemical determinations. Glucose concentrations in tissue culture medium were measured by using a glucose analyzer (model 2300 STAT; YSI Inc., Yellow Springs, Ohio). The detection range for glucose is 0 to
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Confluent Rat Thoracic _ _ . . @ Aorta Smooth Muscle Cells
Feed with 3.0 mls 5 mM ITS/AA --~ medium
Feed with 3.0 mls 5 mM ITS/AA mediumevery 8 hours for 24 hours
Protocol 1 Feed with 3.0 mls 5 or 20 mM ITS/AA medium every 8 hours for 24 hours
Protocol 2 Feed with 3.0 mls __~ 5 o r 2 0 m M 1 0 % FCS medium every 8 hours for 24 hours
Fig. 1. Schematic representation of cell culture methods used in the described experiments to prevent medium glucose depletion. ITS/AA-insulin, transferrin, selenium, and ascorbic acid were at concentrations noted in Methods. FCS, Fetal calf serum.
29.2 mmol/L, with a resolution of 0.1 mmol/L, pH, PCO2, PO2, and HCO3 in tissue culture medium were determined by using a blood gas analyzer (model BG-3; Instrumentation Laboratories, Lexington, Mass.). Medium samples were quickly aspirated from the tissue culture plates and transferred to precooled microcentrifuge tubes that were stored on ice until determinations were performed. Uptake of 2-DG. 2-DG uptake, which measures both glucose transport and phosphorylation, was used as the initial method to examine the glucose transport system in cultured VSMCs. For these uptake experiments, VSMCs were grown in 35 mm dishes. At the indicated time, the medium was aspirated from the cells, and they were washed once with 1 ml of KRH (121 mmol/L NaC1, 4.9 mmol/L KC1, 1.2 mmol/L MgSO4, 0.33 mmol/L CaC12, 12 mmol/L HEPES, pH 7.4) at 37 ° C. A 1 ml sample of uptake solution (KRH with 0.1 mmol/L 2-DG and 1 ixCi/ml tritiated 2-DG) was added to each dish for 3 minutes (uptake was linear for 5 minutes, data not shown) at 37 ° C. The cells were subsequently washed three times with i ml of ice-cold KRH buffer and solubilized with i ml of 1N NaOH. A 500 pA sample of this solution was used for beta scintillation counting, and the remaining 500 txl was neutralized with HC1 and used for protein determination. Five ~mol/L cytochalasin B was included in some uptake experiments to determine nonspecific diffusion, which was subtracted from total cellular uptake. 2-DG uptake measures the combination of transport by the glucose transport protein and phosphorylation by hexokinase. Initial D-glucose transport rate. Initial glucose transport rates were determined by using a modification of the method of Whitesell et al. 26 This method as performed measures only glucose transport and not phosphorylation. At the indicated time, the medium was aspirated and the cells were washed twice with 1 ml 37 ° C KRH containing 0.1 mmol/L glucose and 0.5% BSA. The cells were then incubated at 37 ° C in 600 ixl KRH containing 0.1 mmol/L glucose, 0.5 p~Ci/ml [methyl-14C] 3-O-methylglucose or 0.5 ~Ci/ml [U-~4C] D-glucose and 2 p~Ci/ml [1-3H] L-glucose. For determination of glucose-accessible cell water, cells
were incubated for 1 hour with 3-O-methylglucose, and for determination of initial glucose uptake rates, cells were incubated with D-glucose for 10 seconds. At the end of the incubation period, 2 ml of stop solution (ice-cold KRH containing 200 txmol/L phloretin) was added directly to the uptake solution and rapidly aspirated. Two additional 2 ml washes were performed, and then the cells were solubilized in 1 ml of 0.1N NaOH with shaking for 30 minutes. A 750 Ixl aliquot was used for beta scintillation counting, and the remainder was saved for protein determination. Uptake of carbon 14-labeled glucose was calculated as follows. Raw 14C-labeled glucose space (txl/dish) equals total 14C on the dish after washing (cpm/dish) divided by the concentration of 14C in the uptake solution (cpm/ixl). Raw tritiated L-glucose space 0xl/dish) is calculated as total tritium on the dish after washing (cpm/dish) divided by the concentration of tritium in the uptake solution (cpm/txl). Raw tritiated L-glucose space is then subtracted from the raw 14C-labeled glucose space to yield nonnormalized specific space (~l/disfi). This value is normalized to glucose-accessible intracellular water measured as specific 14C-labeled 3-O-methylglucose space (~l/dish) after a 1-hour incubation. Finally, this value is normalized to protein content per dish, The final units of specific glucose uptake are/~1 of specific glucose space per ixl of glucoseaccessible cell water. Kinetic analysis of glucose transport. These experiments were performed in essentially the same fashion as the initial glucose transport rate experiments, except that the concentrations of glucose utilized were 1, 3, 6, and 10 mmol/L and the a4C-labeled D-glucose was increased to 2 p~Ci/ml to offset lower uptakes with the higher cold glucose concentrations. KM and Vma= were calculated by using a Lineweaver-Burk analysis of the transport data. A minimum of three experiments with an N of 6 at each glucose concentration were performed to calculate the K M and Vm=. Calculation of intracellular glucose concentration. Intracellular glucose concentrations were determined by using a modification of the method of Foley et a l Y At the
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indicated time, the medium was aspirated and the cells were washed twice with 1 ml 37° C KRH containing 0.5% BSA. The cells were then incubated at 37° C for 30 minutes in 600 ixl KRH containing various concentrations of glucose or equiosmotic sucrose, 0.5 ixCi/ml 14C-labeled 3-O-methylglucose, and 2 ixCi/ml tritiated L-glucose. At the end of the 30-minute incubation, 2 ml of stop solution (ice-cold KRH containing 200 ixmol/L phloretin) was added directly to the uptake solution and rapidly aspirated. Two additional 2 ml washes were performed, and then the cells were solubilized in 1 ml of 0.1N NaOH with shaking for 30 minutes. A 750 ~1 aliquot was used for beta scintillation counting, and the remainder was saved for protein determination. These uptake data were used to calculate a ratio of steady-state intracellular 3-O-methylglucose uptake in the presence of glucose to that in the presence of equiosmotic sucrose. With this ratio and the K m for glucose transport, the intracellular glucose concentration (@) can be calculated from the following formula: Ratio = (1 + [Gi]/Km)/ (1 + [Go]/Km), where Gi = intracellular glucose concentration and G o = extracellular glucose concentration. Determination of intracellular glucose concentration with 2-3H-glucose.VSMCs were incubated with tracer
quantities of 2-3H-glucose during the final 8 hours of the 24-hour incubation with 5 or 20 mmol/L glucose. At the indicated time, the medium was aspirated and the cells washed 3 times with 1 ml of ice-cold KRH containing 200 ixmol/L phloretin. The cells were scraped into 1 ml of ice-cold 10 mmol/L Tris-C1 containing 0.5% Triton X-100 and homogenized with a syringe. Tritiated water and unreacted 2-3H-glucose were separated by using a modification of the method described by Hammerstedt. 2s Columns containing Dowex-l-borate were prepared and washed with water. A 500 ixl sample of the homogenized cell sample was applied to the column. Tritiated water was eluted with three 1 ml aliquots of water, and 2-3H-glucose was eluted with three i ml aliquots of 1N HC1. Preliminary experiments revealed that greater than 99% of the tritiated water and less than 1% of the tritiated glucose was recovered in the water elution, and less than 1% of the tritiated water and greater than 99% of the tritiated glucose was recovered in the HCI elution (data not shown). Biotinylation of plasma membranes. Biotinylation of plasma membranes was performed as a modification of the method described by Shetty et al. a9 At the indicated time, the cells were washed twice with 8 ml of ice-cold PBS, and then 1.5 ml of cold biotinylation buffer (120 mmol/L NaC1, 30 mmol/L NaHCO3, 5 mmol/L KC1, pH 8.5) containing 0.1 mg/ml of freshly added NHS-LC-biotin (Pierce, Rockford, Ill.) was added to each dish. The dishes were incubated at 4°C for 30 minutes with occasional swirling. The biotinylation buffer was then aspirated and the dishes were washed three times with 5 ml of buffer containing 140 mmol/L NaC1, 20 mmol/L Tris, and 5 mmol/L KC1 at pH 7.5. Cells from three dishes were then scraped and pooled in 1 ml of hypotonic homogenization buffer containing 10 mmol/L NaHCO 3 and 0.1 mmol/L
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Pefabloc SC (Boehringer Mannheim Corp., Indianapolis, Ind). After 10 minutes on ice, the cells were homogenized with 40 strokes in a glass-Teflon homogenizer. One milliliter of sample was transferred to a microcentrifuge tube, and 100 ~1 of buffer containing 1.5 mol/L NaC1 and 100 mmol/L Tris, pH 7.0, was added. The homogenate was spun for 15 seconds at maximum speed in an Eppendorf Microfuge (Brinkman Instruments, Westbury, N.Y.) to sediment nuclei. The resulting postnuclear supernatant was transferred to a microcentrifuge tube containing 50 ~1 of streptavidin agarose beads (Pierce) that had been sedimented after pre-equilibration with 1 ml of homogenization buffer. After gentle mixing by repeated inversion at 4° C for 30 minutes, the beads were pelleted at 3000 rpm in a refrigerated microcentrifuge at 4°C (International Equipment Co., Needham Heights, Mass.) and washed three times with 1 ml of homogenization buffer containing freshly added Pefabloc SC. The final pellets were resuspended in 120 ixl of ×1.2 Laemmli buffer lacking both mercaptoethanol and bromphenol blue and were incubated at 65 ° C for 30 minutes. The beads were once again pelleted, and the supernatant containing solubilized plasma membrane was removed and frozen. Protein content was determined the next day by using a BCA protein assay kit with BSA and appropriate concentrations of sodium dodecyl sulfate in the standards. Western blot analysis. Cell fractions were denatured by heating for 30 minutes at 60°C in the presence of Laemmli buffer containing 10 mmol/L dithiothreitol. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli, 3° with an 8% acrylamide running gel and a 4% acrylamide stacking gel. Proteins were transferred to nitrocellulose paper (0.2 ~m pore size; Schleicher and Schuell Co., Keene, N.H.) by using a Royal Genie electroblotter (Idea Scientific Co., Minneapolis, Minn.) with a 25 mmol/L Tris, 192 mmol/L glycine, 20% methanol buffer. The nitrocellulose was stained with napthol blue black to assess transfer of proteins. Filters were then blocked overnight with PBS containing 5 % BSA and 0.1% Tween 20. After blocking, filters were washed three times with PBS containing 0.1% Tween 20 and were incubated with a 1:2000 dilution of specific GLUT-1 antibody (East Acres Biologicals, Southbridge, Mass.) in PBS containing 3% BSA and 0.1% Tween 20 for 2 hours. The filter was again washed and the primary antibody detected by incubation with iodine 125-labeled protein A in PBS containing 0.1% Tween 20 for 2 hours. The filters were then washed, dried, and exposed to Kodak XAR-5 film. Band intensities were determined from autoradiograms by using a Seprascan 2001 (Integrated Separations Systems, Hyde Park, Mass.). Estimation of protein content. Total protein content was determined by using the bicinchoninic acid method (BCA Protein Assay Reagent; Pierce) with BSA as the standard. Statistics. Results are reported as the mean + SEM. N equals the number of wells assayed. Statistical analysis was performed by using unpaired Student's t test or analysis of
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150 Cytochalasin B Sensitive 2-Deoxyglucose Uptake (prnole/min/ mg protein)
100
Protocol 2
Protocol 1
Fig. 2. Effect of chronic hyperglycemia on 2-DG uptake in cultured VSMCs. Cells were treated as outlined in Fig. 1, and 2-DG uptake was performed as described in Methods. Open column, 5 mmol/L glucose; hatched column, 20 mmol/L glucose; shadedcolumn, 5 mmol/L glucose plus 15 mmol/L mannitol. N = 6 for each group. *p < 0.01 versus 5 mmol/L glucose group of the same protocol.
Table I. Media parameters obtained by using the two described protocols
Protocol 1 2
Starting glucose concentration Low High Low High
Medium glucose at completion of experiment (mmol/L) 3.5 17.9 1.4 15.2
± 0.1 ± 0,1 ± 0.1 _+ 0.1
pH 7.22 7.23 7.07 7.06
± ± ± +
0.001 0.001 0.005 0.009
Pco2 (torr) 41.8 39.7 51.2 50.5
± 1.1 ± 0,8 ± 0.9 _+ 0.6
P2 (torr) 129 144 115 122
± ± ± +
6.8 5.7 2.7 1.6
HC03 (mmol/L)
17.3 ± 0.2 16.6 ± 0.1 14.8 ± 0.2 14.5 + 0.3
Cultured rat aortic smooth muscle cells were treated as described in Methods and in Fig. 1. N = 6 for all groups.
variance, as indicated in the Results section, andp values less than 0.05 were considered significant. To investigate whether there was a significant difference in the cytochalasin B-sensitive uptake of 2-DG in VSMCs, a two-way analysis of variance with interaction was computed. Specifically, if the interaction is significant, then there exists evidence of a difference in the cytochalasin B-sensitive uptake of 2-DG between the various extraceIlular glucose concentration groups. RESULTS Cell culture methods. Previous investigations have noted that freshly isolated porcine arteries exhibit high rates of aerobic glycolysis.24'25 Cultured rat aortic smooth muscle cells exhibited similar characteristics, resulting in rapid medium glucose utilization and lactate production (data not shown). To avoid medium glucose depletion, which up-regulates the glucose transport system, the cell culture and feeding protocol outlined in Fig. 1 was utilized for all experiments. Medium was changed every 8 hours
to prevent medium glucose depletion and to maintain a more constant pH. As shown in Table I, the most physiologic medium conditions were obtained when cells were cultured in the serum-free (ITS/ AA) medium. When serum was added to the medium, glucose utilization increased and subsequent lactate production increased, resulting in lower medium glucose and pH values. Uptake of 2-DG. As an initial method to examine the chronic effect of elevated extracellular glucose concentrations on the glucose transport system, we utilized the 2-DG uptake assay, which measures both glucose transport and phosphorylation. As shown in Fig. 2, 2-DG uptake was significantly decreased (two-way analysis of variance with interaction) in VSMCs exposed to elevated extracellular glucose concentrations for 24 hours. The percent decrease in 2-DG uptake was similar in the presence or absence of serum (approximately a 36% decrease), and it can be seen that the rate of 2-DG uptake was higher in the cells exposed to serum.
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Fig. 3. Effect of chronic hyperglycemia on initial D-glucose uptake rate in cultured VSMCs. Cells were treated as outlined in Fig. 1, and D-glucose uptake was performed as described in Methods. A, Protocol 1. B, Protocol 2. Open column, 5 mmol/L glucose; hatched column, 20 mmol/L glucose; shaded column, 5 mmol/L glucose plus 15 mmol/L mannitol. N = 6 for each group in each experiment. *p < 0.01 versus 5 mmol/L glucose group of the same experiment.
These results suggest that either a decrease in glucose transport or a decrease in glucose phosphorylation occurs in the VSMCs exposed chronically to hyperglycemic medium. Initial D-glucose transport rate. To investigate whether the alterations in 2-DG uptake were the result of changes in glucose transport or phosphorylation, we utilized the initial D-glucose transport assay. By utilizing the natural substrate for the glucose transporter (D-glucose) and short uptake periods (10 seconds), we were able to directly estimate the glucose transport parameters. As shown in Fig.
3, the initial D-glucose transport rate was significantly decreased (Student's t test) in VSMCs exposed to hyperglycemic medium for 24 hours. Again, it can be seen that glucose transport was higher in the cells exposed to serum and that the decrease in glucose transport occurred in the presence or absence of serum. These results suggest that a decrease in glucose transport occurs in VSMCs exposed chronically to hyperglycemic medium. Mechanism of decreased glucose transport. We next set out to determine the mechanism responsible for the decrease in glucose transport in VSMCs exposed
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GLUT - 1
45,000
31,000 0.955__+0.12
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Fig. 4. Effect of chronic hyperglycemia on GLUT-1 transport protein in plasma membrane fraction. Cells were treated as outlined in protocol 1 of Fig. 1, and plasma membrane proteins were biotinylated, immunoprecipitated, and immunoblotted for the GLUT-1 protein as described in Methods. A 10 ~g sample of plasma membrane protein from three pooled 100 mm plates was run in each lane. The four lanes in each group represent separate experiments on different passages of cells. Molecular weight markers are indicated on the right side of the figure. The numbers below the autoradiograph represent the arbitrary densitometry units for each group (p value < 0.05).
Table II. Kinetic analysis of glucose transport in culfured VSMCs Protocol 1 2
Glucose concentration 5 20 5 20
mmol/L mmol/L mmol/L mmol/L
Km (retool/L) 1.74 1.95 2.86 2.77
± 0.12 --- 0.14 ± 0.08 --- 0.10
Vma x
p value NS NS
(pmol/sec/mg protein) 101 53 426 222
± 2.0 _+ 1.7 ± 8.7 -+ 6.4
p value <0.01 <0.01
Cultured rat aortic smooth muscle ceils were treated as described in Methods and in Fig, 1, Kinetic analysis was performed as described in Methods. The results are pooled from three separate passages of ceils with N = 6 for each group in each passage.
chronically to hyperglycemic medium. As shown in Table II, kinetic analysis of glucose transport revealed a significant decrease in the Vma x for glucose transport without a significant change in the K m for glucose transport in VSMCs exposed to hyperglycemic medium for 24 hours. The kinetic changes in glucose transport were similar in the presence or absence of serum, and the Km and Vmax for glucose transport were higher in the VSMCs exposed to serum for 24 hours. This decrease in Vmax could be the result of either a decrease in the absolute number of glucose transporters or a decrease in the transport rate of the glucose transporters present at the plasma membrane. To examine this question, we used biotinylation of surface membrane proteins followed by immunoprecipitation of the labeled proteins and immunoblotting of the GLUT-1 transport protein as described by Shetty et al.29 The results of these experiments are shown in Fig. 4. The figure shows that the quantity of GLUT-1 transport protein present at the plasma membrane is decreased in
VSMCs exposed to hyperglycemic medium for 24 hours. Densitometry measurements were performed on the blot in Fig. 4; the results are shown below the figure, and they confirm a decrease in plasma membrane GLUT-1 transport protein in VSMCs exposed chronically to hyperglycemic medium. Effect of alterations in glucose transport on intracellulor
glucose concentrations. The final issue that we wanted to examine concerned the effect of decreased glucose transport in VSMCs exposed chronically to hyperglycemic medium on intracellular glucose concentrations. To examine this question, we used two methods to estimate intracellular glucose concentrations. The first method utilized steady-state countertransport kinetics to measure the intracellular glucose concentration indirectly.27 As shown in Fig. 5, A, the ratio of steady-state intracellular 14C-labeled 3-O-methylglucose in the presence of glucose to that in the presence of equiosmotic sucrose is lower at both buffer glucose concentrations used in VSMCs exposed to hyperglycemic medium in the absence of
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Fig. 5. Effect of chronic hyperglycemia on indirectly determined intracellular glucose concentration. Cells were treated as outlined in protocol 1 of Fig. 1, and steady-state intracellular 3-O-methylglucose concentrations were determined as described in Methods. A, Ratio of intracellular 3-O-methylglucose in the presence of the indicated buffer glucose concentrations or equiosmotic sucrose. B, Intracellular glucose concentration calculated from the ratios obtained in A and the I ~ values from Table II. The results were obtained from three experiments with N = 3 for each experiment. *p < 0.01 versus same medium glucose concentration and 5 mmol/L buffer glucose concentration. #p < 0.01 versus 5 mmol/L medium glucose concentration and 5 mmol/L buffer glucose concentration.
s e r u m for 24 hours. T h e lower ratio in the V S M C s e x p o s e d to chronically e l e v a t e d extracellular glucose c o n c e n t r a t i o n s (at e i t h e r buffer glucose c o n c e n t r a tion) is consistent with the d e c r e a s e d glucose transp o r t n o t e d in e a r l i e r e x p e r i m e n t s . T h e c o r r e s p o n d -
ing c a l c u l a t e d i n t r a c e l l u l a r glucose c o n c e n t r a t i o n s a r e shown in Fig. 5, B, a n d r e v e a l that in the face of d e c r e a s e d glucose t r a n s p o r t in V S M C s e x p o s e d chronically to h y p e r g l y c e m i c m e d i u m , the intracellular glucose c o n c e n t r a t i o n r e m a i n s h i g h e r t h a n in
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Fig. 6. Effect of chronic hyperglycemia on indirectly determined intracellular glucose concentration. Cells were treated as outlined in protocoI 2 of Fig. 1, and steady-state intracellular 3-O-methylglucose concentrations were determined as described in Methods. A, Ratio of intracellular 3-O-methylgtucose in the presence of the indicated buffer glucose concentrations or equiosmotic sucrose. B, Intracellular glucose concentration calculated from the ratios obtained in A and the K~ values from Table II. The results were obtained from three experiments with N = 3 for each experiment. *p < 0.01 versus same medium glucose concentration and 5 mmol/L buffer glucose concentration. # p < 0.01 versus 5 mmol/L medium glucose concentration and 5 mmol/L buffer glucose concentration.
cells exposed to normoglycemic medium (compare the bars labeled 5/5 and 20/20, which represent the actual conditions to which the cells were exposed). Fig. 6 shows that similar results were seen in cells incubated in the presence of serum, although the degree of down-regulation of intracellular glucose concentration appears to be less dramatic. When Figs. 5 and 6 are compared, it can be noted that intracellular glucose concentrations are significantly higher in cells incubated in the presence of serum. A second method for estimating intracellular glucose was also used. This method utilizes tritiated
glucose as a tracer, and it requires separation of the tritiated glucose from tritiated water, generated in the metabolism of glucose-6-phosphate to fructose6-phosphate, by using Dowex chromatography. As shown in Fig. 7, VSMCs exposed to hyperglycemic medium for 24 hours exhibited significantly higher (Student's t test) intracellular glucose concentrations than cells exposed to normoglycemic medium. As noted for the first method, intracellular glucose concentrations were higher in cells exposed to serum, and the higher intracellular glucose concentrations seen with hyperglycemic culture conditions oc-
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Fig. 7. Effect of chronic hyperglycemia on intracellular glucose concentration determined by using tritiated glucose. Cells were treated as outlined in Fig. 1, and intracellular tritiated glucose concentrations were determined as described in Methods. Open column, 5 mmol/L glucose; hatched column, 20 mmol/L glucose. N = 6 for each group in each experiment. *p < 0.01 versus 5 mmol/L glucose group of the same protocol.
curred in the presence or absence of serum. The results from these two sets of studies suggest that intracellular glucose concentrations remain higher in VSMCs exposed chronically to hyperglycemic medium in spite of decreased glucose transport. DISCUSSION
In the present study we have shown that chronic elevation of extracellular glucose concentration (20 mmol/L glucose for 24 hours) leads to down-regulation of glucose transport by decreasing the number of GLUT-1 glucose transporters present at the plasma membrane. In addition , we have utilized two separate methods to show that this down-regulation of glucose transport does not result in normalization of intracellular glucose concentration after a 24hour exposure to elevated extracellular glucose concentrations. These results are similar to those reported by Kaiser et al} 4 but provide additional important information regarding the regulation of glucose transport in cultured VSMCs. Kaiser et al. reported a decrease in glucose transport and crude membrane GLUT-1 protein in bovine and human smooth muscle cells exposed to hyperglycemic medium for 24 hours. Cells exposed to the two lowest medium glucose concentrations (1.2 and 5.5 mmol/L) in this study may have been exposed to depleted or neardepleted medium glucose conditions at the time the assays of glucose transport were performed because of the culture protocol used. If this situation occurred, resulting in up-regulation of glucose trans-
port caused by medium glucose depletion, the results would be difficult to interpret. In contrast, we have utilized a culture protocol designed specifically to prevent medium glucose depletion and to provide variation in medium glucose concentrations and pH over a narrow range (see Fig. 1 and Table I). Using the described cell culture protocol, we have also found that chronic elevation of extracellular glucose concentrations results in down-regulation of glucose transport as measured by both 2-DG uptake (Fig. 2) and initial D-glucose transport rates (Fig. 3). While 2-DG uptake is technically easier because of the prolonged uptake periods, it measures both transport and phosphorylation of the non-metabolizable 2-DG. Initial D-glucose transport reflects only the transport of glucose when short time points are used (10 seconds in this study). It should be noted that the down-regulation of glucose transport was much more dramatic in Kaiser's studies, and again this may reflect the culture protocol used. Similar to Kaiser et al., 14 we have also found that the decrease in glucose transport is the result of a decrease in Vm~x for glucose transport (Table II). Of note, our calculated K~ values for glucose transport are significantly higher than those reported by Kaiser et al. (1.75 to 1.95 mmol/L versus approximately 0.85 mmol/L). We believe these differences reflect the different methods used. Kaiser et al. performed kinetic analysis of glucose transport by using 2-DG uptake, whereas we utilized initial D-glucose uptake rates. We chose to use D-glucose because it is the natural substrate for the glucose transporter and
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therefore does not perturb intracellular metabolism. In contrast, 2-DG can be transported and phosphorylated but not metabolized further. Using a recently developed technique to label surface membrane proteins with biotin followed by immunoprecipitation and immunoblotting for the GLUT-1 protein, we have been able to show that the decreased Vm~x of glucose transport is associated with a decrease in the quantity of GLUT-1 glucose transporter at the plasma membrane (Fig. 4). Kaiser et al. were unable to find a difference in the quantity of GLUT-1 transport protein in a crude membrane fraction when comparing cells exposed to 5.5 and 22 mmol/L glucose. Kaiser et al. 14 suggest that "the ability of VSMCs to down-regulate glucose transport in response to chronic hyperglycemia may serve as a protective mechanism against possible adverse effects of increased intracellular glucose." We are not aware of any measurements or estimates of intracellular glucose in VSMCs that actually address this issue. We have used two methods to indirectly (steady-state intracellular 3-O-methylglucose concentrations) and directly (tritiated glucose) estimate intracellular glucose concentrations. Although the two methods yielded different results regarding the magnitude of the differences, both methods revealed that intracellular glucose concentrations were significantly elevated in VSMCs exposed to chronic elevations of extracellular glucose concentrations when compared with cells exposed to normal extracellular glucose concentrations (Figs. 5 through 7). Although the intracellular concentration of glucose that is "toxic" to the cell has not been documented, and although we have no measurements of potentially toxic glucose metabolites (sorbitol, myoinositol, amphipathic lipids, protein kinase C) to document toxicity in our system, we believe that the potential for toxicity related to increased intracellular glucose concentrations remains even after 24 hours of elevated extracellular glucose concentrations. This inability of VSMCs to normalize intracellular glucose concentrations, resulting in prolonged increases in intracellular glucose concentrations, may explain why the vascular system is so prominently affected in patients with diabetes mellitus and hyperglycemia. Another interpretation of these data would be that the elevated intracellular glucose concentrations result from decreased metabolism of glucose to toxic metabolites. This response would protect the cell from toxic glucose metabolites but raise the intracellular glucose concentration. Detailed measurements of glucose metabolites will be required to clarify this issue. The presence of measurable intracellular concen-
trations of free glucose in all experimental conditions used indicates that glucose metabolism and not glucose transport is the rate-limiting step for glucose uptake in cultured smooth muscle cells. Clarification of the changes in glucose metabolism associated with elevated extracellular glucose concentrations may provide insight into the vascular disease associated with diabetes mellitus and hyperglycemia. In summary, we have shown that exposure of cultured VSMCs to chronically elevated extracellular glucose concentrations results in down-regulation of glucose transport that is the result of a decreased quantity of GLUT-1 glucose transport protein at the plasma membrane. This down-regulation of glucose transport does not result in normalization of intraceUular glucose concentration after a 24-hour exposure to elevated extracellular glucose concentrations. Down-regulation of glucose transport does not appear to normalize intracellular glucose concentrations over the time period studied, and these results may explain the susceptibility of VSMCs to the toxic effects of hyperglycemia. I thank Daniel J, Homco, BS, for technical assistance. REFERENCES
1. Viberti GC, Messent J. Introduction: hypertension and diabetes. Critical combination for micro- and macrovascular disease. Diabetes Care 1991;14(suppl 4):4-7. 2. Jarrett RJ, Shipley MJ. Mortality and associated risk factors in diabetics. Acta Endocrinol 1985;110(suppl 272):21-6. 3. Jarret RJ, Keen H, Chakrokati A. Diabetes, hyperglycemia and arterial disease. In: Keen H, Jarret RJ, eds. Complications of diabetes. London: Edward Arnold, 1982:79-84. 4. Krolewski AS, Warren JH, Rand LI, Kahn CR. Epidemiological approach to the etiology of type 1 diabetes and its complications. N Engl J Med 1987;317:1390-8. 5. Pirart J. Diabetes mellitus and its degenerative complications: a prospective study of 4400 patients observed between 1947-1973. Diabetes Care 1978;1:168-88. 6. The DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977-86. 7. Pugliese G, Tilton RG, Williamson JR. Glucose-induced metabolic imbalances in the pathogenesis of diabetic vascular disease. Diabetes Metab Rev 1991;7:35-59. 8. Wilson PW, Cupples LA, Kannel WB. Is hyperglycemia associated with cardiovascular disease? The Framingham study. Am Heart J 199I;121:586-90. 9. Pell S, D'Aionzo CA. Some aspects of hypertension in diabetes mellitus. JAMA 1967;202:104-10. 10. Jarrett RJ, Keen H, McCartney M, et al. Glucose tolerance and blood pressure in two population samples: their relation to diabetes mellitus and hypertension. Int J Epidemiol 1978; 7:15-24. 11. Christlieb AR, Warram JH, Krolewski AS, et al. Hypertension: the major risk in juvenile-onset insulin-dependent diabetics. Diabetes 1981;30(suppl 2):90-6.
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