Glucose transporters control gene expression of aldose reductase, PKCalpha , and GLUT1 in mesangial cells in vitro

Douglas N. Henry1,2, Julia V. Busik1, Frank C. Brosius III3, and Charles W. Heilig4

1 Department of Physiology, 2 Department of Pediatrics and Human Development, Division of Pediatric Endocrinology, College of Human Medicine, Michigan State University, East Lansing 48824-1101; 3 Department of Medicine, Division of Nephrology, University of Michigan Medical School and Ann Arbor Veterans Affairs Hospital, Ann Arbor, Michigan 48109; and 4 Department of Medicine, Division of Nephrology, University of Rochester School of Medicine, Rochester, New York 14642


    ABSTRACT
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INTRODUCTION
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The process linking increased glucose utilization and activation of metabolic pathways leading to end-organ damage from diabetes is not known. We have previously described rat mesangial cells that were transduced to constitutively express the facilitative glucose transporter 1 (GLUT1, MCGT1 cells) or bacterial beta -galactosidase (MCLacZ, control cells). Glucose transport was rate limiting for extracellular matrix production in the MCGT1 cells. In the present work, we investigated the effect of GLUT1 overexpression in mesangial cells on aldose reductase (AR), protein kinase Calpha (PKCalpha ), and native GLUT1 transcript levels, to determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. MCGT1 cells grown in normal (8 mM) or elevated (20 mM) glucose had elevated abundance of AR, PKCalpha , and the native GLUT1 transcripts compared with control cells. AR protein levels, AR activity, sorbitol production, and PKCalpha protein content were also greater in the MCGT1 cells than in control cells grown in the same media. This is the first report of the concomitant activation of AR, PKCalpha , and GLUT1 genes by enhanced GLUT1 expression. We conclude that increased GLUT1 expression leads to a positive feedback of greater GLUT1 expression, increased AR expression and activity with polyol accumulation, and increased total and active PKCalpha protein levels, which leads to detrimental stimulation of matrix protein synthesis by diabetic mesangial cells.

gene regulation; facilitative glucose transport; diabetic nephropathy; genetics; hyperglycemia


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

OVER THE PAST SEVERAL YEARS, reports have documented the process linking increased glucose utilization and stimulating of extracellular matrix (ECM) production by renal mesangial cells (RMC). These findings have reinforced the important role of glucose in stimulating mesangial cell ECM production, which in vivo would lead to glomerulosclerosis in diabetes. It has long been assumed that by unknown mechanisms, high extracellular glucose concentrations lead to increased glucose uptake into the mesangial cell to stimulate a processes that culminate in increased ECM accumulation and glomerulosclerosis. Recently, three facilitative glucose transporters were identified in RMC (8, 9, 30, 59). All three transporters are low-Km (Km of 1-7 mM), high-affinity transporters for glucose, which would be at or near saturation at physiological glucose concentrations. Therefore, we recently investigated the mechanism by which high extracellular glucose leads to increased intracellular glucose availability in RMC (29, 35). We found that extracellular glucose in the diabetic range (20 mM) is a potent stimulus to increase mesangial cell facilitative glucose transporter 1 (GLUT1) expression (29). Furthermore, it leads to increased glucose uptake and enhances insulin-like growth factor I (IGF-I)-induced glucose uptake in RMC (29). The increase in mesangial cell GLUT1 is maintained even after 3 mo in 20 mM high glucose (29). Therefore, a detrimental positive feedback mechanism exists in RMC, whereby high extracellular glucose may stimulate its own uptake and induce the diabetic mesangial cell phenotype. The potential for GLUT1 to regulate mesangial cell glucose uptake, and by implication the downstream pathways leading to ECM synthesis, was thereby recognized (31).

To examine the role of GLUT1 in regulating mesangial cell ECM synthesis, we subsequently overexpressed GLUT1 in rat RMC (MCGT1) or beta -galactosidase as a control (MCLacZ) (35). MCGT1 cells demonstrated a fivefold increase in glucose uptake and greater synthesis of individual ECM components than the MCLacZ control cell (35). This work clearly demonstrated a role for GLUT1 in regulating mesangial cell ECM gene expression and synthesis. By developing a stable transduced mesangial cell line overexpressing the human GLUT1 facilitative transporter, we demonstrated that high extracellular glucose was not required for increased glucose uptake or for accumulation of sorbitol, lactate, and ECM synthesis (35). It was also clear that hyperosmolality was not required for these events to take place.

Previous work by other investigators demonstrated what was believed to be hyperosmolar induction of the aldose reductase (AR) gene by high extracellular NaCl or glucose concentrations (33, 50). A specific role for glucose transport or metabolism was not examined. Currently, much effort is being expended to identify osmotic response elements in the AR gene (6, 21); however, no data exist concerning the potential existence of a glucose responsive element (GlRE) in this gene. Protein kinase C (PKC) is activated in diabetic glomeruli and glucose-treated mesangial cells, which may direct the transcription of ECM genes (4, 16, 37). No work to our knowledge has examined the PKCalpha or GLUT1 genes for GlREs.

Therefore, in this work we investigated the effect of GLUT1 overexpression in RMC on AR, PKCalpha , and GLUT1 transcript levels to determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. We found the transcript levels for AR, PKCalpha , and GLUT1 were all substantially increased in MCGT1 cells vs. the MCLacZ control cells. Furthermore, AR activity and protein were significantly elevated under normal and elevated glucose conditions, corresponding with increased sorbitol levels. MCGT1 cells grown in normal (8 mM) or pathophysiological (20 mM) levels of glucose had high expression of AR. These results confirmed an important role for GLUT1 in regulating the expression of these genes, separate from high extracellular glucose exposure and hyperosmolality. The results further imply the presence of a GlRE or transcript stabilization mechanisms activated by increased expression of GLUT1. The GLUT1-regulated pathway of glucose transporters, polyol pathway, and the PKCalpha pathway activation likely contribute to the enhanced ECM synthesis characteristic of GLUT1-overexpressing RMC and potentially to the development of mesangial scarring in diabetes.


    METHODS
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Reagents and supplies. Unless otherwise noted, culture media, trypsin, antibiotics, and Hanks' PBS were obtained from GIBCO-BRL (Life Technologies, Gaithersburg, MD); culture dishes, wells, and flasks were from Falcon (Becton-Dickinson, Lincoln Park, NJ); chemicals and reagents (highest purity available) were from Sigma Chemical (St. Louis, MO); radioisotopes were from New England Nuclear Research Products (DuPont, Wilmington, DE); and NuSerum was from Collaborative Research (Bedford, MA).

Tissue culture. Rat RMC transduced to overexpress human GLUT1 transporter (MCGT1 cells) or beta -galactosidase (MCLacZ cells) were used in these experiments (35). Transduction of these cells was previously described (35) using a pWZLneo retroviral vector (Ariad Pharmaceutical, Cambridge, MA) that contains an internal ribosomal entry site from the encephalomyelocarditis virus, which allows for translation of the GLUT1 and neomycin phosphotransferase (neor) gene products from the same RNA transcript. A control construct containing the bacterial beta -galactosidase gene (LacZ) but lacking the GLUT1 gene was used to create the MCLacZ control cells (35). The cells were grown in RPMI-1640 media with 8 mM glucose and 2.05 mM glutamine, supplemented with folic acid (225 µM), 1% penicillin/streptomycin, 20% NuSerum, and 250 µg/ml of G418 (Geneticin, GIBCO-BRL). Cells were grown in 95% humidified air with 5% CO2 at 37°C. MCLacZ and MCGT1 cells were seeded at 10,000 cells/cm2 in 10-cm plastic tissue culture dishes in media containing 8 mM glucose and allowed to grow for 48 h prior to changing media to experimental conditions (8 mM normal glucose and 20 mM elevated glucose). Media was changed every other day without a significant decline in glucose concentration (data not shown). Experiments were terminated after 7 days of growth in experimental conditions when the cells reached confluence.

Northern analysis. Total RNA was isolated using a modification of the acid phenol extraction method (13). This procedure yields ~100 µg of total RNA from confluent monolayers of RMC grown in 10-cm plates. A quantity of 10 µg of total RNA was resolved on denaturing 2.2 M formaldehyde-1% agarose gels and transferred to ZetaBind (Cuno, Meriden, CN) nylon filters by capillary blotting. The filter was stained with methylene blue to examine the integrity of the RNA and to assess the uniformity of loading and transfer. The filters were fixed by ultraviolet cross-linking and prehybridized at 55°C in 0.5 M NaH2PO4, pH 7.0, 1 M EDTA, 7% SDS, and 1% BSA for 4-6 h before adding the denatured cDNA probes according to the protocol of Church and Gilbert (15). Probes were labeled with [alpha -32P]dCTP (3,000 Ci/mmol) using random primers to a specific activity of 109 dpm/mg and separated from unincorporated nucleotides by gel filtration (20). After 18 h, filters were washed twice with 40 mM NaH2PO4, pH 7.0, 1 mM EDTA, and 1% SDS at 55°C. Autoradiograms were obtained using multiple exposures to remain within the linear range of the film (X-Omat; Eastman-Kodak, Rochester, NY). Each blot was hybridized concomitantly with a human AR (33), GLUT1 (45), and rat PKCalpha (48) cDNA probes. Filters were stripped until free of radioactivity and were checked by autoradiography prior to rehybridization with a rat beta -tubulin cDNA probe (53). Optical-scanning densitometry (AGFA; AGFA-Gevnert, Mortsel, Belgium), high-resolution optical scanner, and NIH Image gel plotting software (46) were used to analyze the autoradiograms.

Measurement of AR activity and sorbitol content. AR protein activity was measured spectrophotometrically at 340 nm using 10 mM DL-glyceraldehyde as substrate and expressed as the amount of NADPH that was oxidized per minute per milligram protein (51) at 37°C using a temperature-controlled spectrophotometer (Beckman DU model 640) with a modification of the method of Das and Srivastava (17). Sorbitol levels were determined spectrophotometrically at 340 nm, at room temperature, using NAD+ as substrate and sorbitol dehydrogenase (7). Samples were deproteinized using 6% perchloric acid prior to assay. Standard curves of sorbitol concentrations in the sorbitol dehydrogenase reaction were performed for each determination, and results normalized to total cellular protein content (51).

Immunoblotting of AR. Immunoblots of AR protein were performed according to the methods previously described (1). We mixed 300-µl aliquots of RMC cell homogenate with 150 µl of SDS-containing sample buffer, heated at 95°C for 5 min, mixed with 50 µl of 20% beta -mercaptoethanol, and stored at -20°C. Following thawing, samples corresponding to 10 µg of cell protein per lane were electrophoresed on 7.5% linear polyacrylamide minigels. The separated proteins were then transferred to nitrocellulose sheets by conventional procedures. The transferred AR protein was visualized by incubation for 2 h at 23°C in PBS containing a 1:400 dilution of the primary rat anti-AR antibody, followed by a 90-min incubation at 23°C in PBS containing a 1:400 dilution of a commercial immunoglobulin-peroxidase conjugate (Sigma Chemical). Following development for peroxidase reactivity, scanning densitometry with a high-resolution AGFA optical scanner and NIH Image software were used to quantitate blots. Identification of AR was confirmed by analysis of the molecular weights and comparison with previously reported molecular weights (1).

Immunoblotting of PKCalpha . Immunoblot analysis was carried out according to methods previously described with minor modification (43). Solubilized protein samples of 50 µg were separated by SDS-PAGE and electrophoretically transferred to Hybond-ECL nitrocellulose membranes (Amersham). As primary antibodies, PKCalpha , PKCbeta 1, PKCdelta , and PKCzeta polyclonal antibodies were used (43). The secondary antibody was a horseradish peroxidase anti-rabbit Ig conjugate (Amersham). Immunoblotting of beta -tubulin or Ponceau-S staining was used as a confirmatory method to assure equal sample loading between gel lanes. Identification of PKC isoforms was confirmed by analysis of the molecular weights and comparison with previously reported molecular weights.

Statistical analysis. Results are expressed as means ± SE of at least four experiments except where noted. Statistical significance of differences between experimental groups was determined using ANOVA, Student's t-test, or the Bonferroni correction of t-test. Other statistical methods were used where noted.


    RESULTS
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Induction of AR, PKCalpha , and native GLUT1 mRNAs in RMCs in vitro. AR is the rate-limiting enzyme of polyol metabolism and has been implicated in the development of diabetic complications in retinal, renal, nerve, and vascular smooth muscle tissue. As noted above, both facilitative glucose transporters and PKC have been reported to play a key role in the development of mesangial cell ECM synthesis in diabetes. Transcripts for each of these proteins can be resolved by northern analysis because of their differences in size (Fig. 1; AR, 1.8 kb; native GLUT1, 2.8 kb; and PKCalpha , 4.3 kb). The proviral human GLUT1 transcript was constitutively expressed at a high level in the MCGT1 cells, which can be identified by the larger size of the proviral transcript (5.6 kb) compared with the native GLUT1 transcript (2.8 kb). Concomitant increases in the abundance of AR, native (rat) GLUT1, and PKCalpha mRNA were conferred by increased (human) GLUT1 mRNA expression in the MCGT1 cells grown in all glucose concentrations vs. the MCLacZ control cells grown in the same conditions.



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Fig. 1.   Concomitant activation of aldose reductase (AR), protein kinase Calpha (PKCalpha ), and native facilitative glucose transporter 1 (GLUT1) in renal mesangial cell (RMC) that overexpress the GLUT1 transporter (MCGT1 cells). A: Northern blot of 10 µg of total RMC RNA. Blot was then concomitantly hybridized with cDNAs for GLUT1, PKCalpha , and AR. Blot was then stripped and reprobed with beta -tubulin to determine relative loading of total RNA. Control cells are RMCs transduced to overexpress the bacterial beta -galactosidase gene (MCLacZ). Cells were grown for 4 days in media containing 8 mM and 20 mM glucose. B: quantitation of AR mRNA expression in RMC from Northern blots. Data for AR (solid bar), GLUT1 (hatched bar), and PKCalpha (open bar) were normalized to beta -tubulin mRNA expression. * P < 0.05 for MCGT1 vs. MCLacZ in 8 mM glucose. # P < 0.05 for MCGT1 vs. MCLacZ in 20 mM glucose. Measured transcripts were significantly (P < 0.05) greater for MCGT1 vs. MCLacZ in all growth conditions. Results are means ± SE of 4 experiments.

Although high glucose transport concomitantly increased AR, PKCalpha , and native GLUT1 transcript levels in the MCGT1 cells, the control MCLacZ cells also demonstrated increases in each of these transcripts when exposed to increases in glucose concentration in the growth media. MCLacZ AR mRNA increased 2.1-fold when grown in 20 mM vs. 8 mM glucose. GLUT1 transcript abundance also increased in MCLacZ cells grown in media containing 20 mM vs. 8 mM glucose. The PKCalpha transcript was detected at the limits of sensitivity by Northern analysis when the MCLacZ cells were grown in 8 mM glucose.

Effect of increased glucose transport on AR protein expression, AR enzyme activity, and sorbitol content. To further understand the effects of increased glucose transport on activation of polyol metabolism in the RMC cells, we quantitated changes in AR protein content and activity in MCGT1 and MCLacZ cells grown in media containing 8 or 20 mM glucose. The increase in the abundance of AR mRNA was accompanied by increased AR protein expression (Fig. 2) and enzyme activity (Fig. 3) in the RMC cells.


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Fig. 2.   Expression of AR protein in RMC cells. A: immunoblot of AR protein from mesangial cells grown in 8 mM and 20 mM glucose. MCLacZ cells (left) and MCGT1 cells (right). AR protein was detected at ~39 kDa. B: quantitation of AR protein expression from 3 experiments. MCLacZ cells (open bars), MCGT1 cells (solid bars). * P < 0.05 for 20 mM vs. 8 mM glucose. AR protein was significantly (P < 0.05) greater for MCGT1 vs. MCLacZ in all growth conditions. Single statistic t-test, n = 4 experiments.



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Fig. 3.   AR activity in RMC. AR enzyme protein activities were measured in MCLacZ (open bars) and MCGT1 (solid bars) cells grown in 8 mM or 20 mM glucose. * P < 0.05 for 20 mM vs. 8 mM glucose. Measured AR activities were significantly (P < 0.05) greater for MCGT1 vs. MCLacZ in all growth conditions. Results are means ± SE of 4 experiments.

The metabolic correlates of AR gene expression were assessed by comparing the dose-dependent changes in sorbitol content after exposure to normal (8 mM) or hyperglycemic concentrations of glucose (20 mM) in MCGT1 and MCLacZ cells (Fig. 4). MCGT1 cells had greater abundance of sorbitol than the MCLacZ cells when grown in both 8 mM glucose (68% sorbitol increase) and 20 mM glucose (60% sorbitol increase). Although both RMC lines demonstrated increased AR protein content and activity in high glucose, the MCGT1 cells had greater levels of AR protein and higher AR activity than did MCLacZ cells grown in the same glucose concentrations.


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Fig. 4.   Sorbitol content in RMC. Sorbitol levels (nmol/mg protein) were determined in MCLacZ (open bars) and MCGT1 (solid bars) after 4 days of growth in media containing 8 mM or 20 mM glucose. * P < 0.05 for 20 mM vs. 8 mM glucose. Measured sorbitol contents were significantly (P < 0.05) greater for MCGT1 vs. MCLacZ in all growth conditions. Results are means ± SE of 4 experiments.

Effect of increased glucose transport on PKC expression. Quantitative immunoblotting of PKC isoforms was conducted to determine whether enhanced glucose transport alone enhanced PKC expression. Total PKCalpha protein was 40% more abundant in the MCGT1 cells than in the MCLacZ cells (Fig. 5). Total PKCdelta and PKCzeta protein levels were not different in the MCGT1 and MCLacZ cells (Fig. 5). Immunoblot analyses of PKCalpha protein in MCLacZ and MCGT1 cells were also performed after separation of proteins into their cytosolic and particulate membrane fractions (Fig. 6). Quantitation of PKCalpha cytosolic and particulate (active) membrane fractions demonstrated a 30% greater cytosolic and 60% particulate (active) PKCalpha fraction in the MCGT1 than in the MCLacZ cells when RMC were grown in media containing 8 mM glucose (Fig. 7).


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Fig. 5.   Expression of PKCalpha , -delta , and -zeta proteins in transduced RMC (MCLacZ and MCGT1 cells) grown in 8 mM glucose medium. Total PKCalpha , -delta , and -zeta isoform proteins are shown in MCGT1 cells (solid bars with SE) compared with their levels in control MCLacZ cells (note line at 100%). Levels of individual PKC proteins determined by densitometric analysis are shown for MCGT1 cells (solid bars) plotted as percent of control (MCLacZ) (error lines are SE). * P < 0.05 for PKCalpha in MCGT1 vs. MCLacZ cells, single statistic t-test, n = 4 experiments.



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Fig. 6.   Immunoblot of PKCalpha protein in MCLacZ and MCGT1 cells grown in 8 mM glucose. Cytosolic (Cyto) and particulate membrane (PM) fractions were isolated from the 2 cell types. PKCalpha protein was identified on immunoblots with specific antibody, at ~80 kDa.



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Fig. 7.   Semiquantitation of PKCalpha protein in cytosolic and particulate membrane fractions of MCLacZ and MCGT1 cells. MCLacZ and MCGT1 cells were both grown in 8 mM glucose medium. PKCalpha protein was detected at ~80 kDa with specific antibody. Levels of PKCalpha protein in each cell fraction were determined by densitometric analysis, and levels in MCGT1 cells were plotted as percent control (+1 SE) vs. levels in control MCLacZ cells (100%). * P < 0.05 (single statistic t-test) for MCGT1 vs. MCLacZ cells in both the cytosolic (hatched bar) and particulate membrane (solid bar) fractions; n = 5 experiments.


    DISCUSSION
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INTRODUCTION
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This is the first report of concomitant activation of AR, PKCalpha , and GLUT1 genes from excessive transport of glucose in the absence of high extracellular glucose or osmolality. RMCs overutilizing glucose concomitantly activate genes believed to contribute to the metabolic disturbances that promote the development of diabetic nephropathy.

Excessive ECM synthesis and release by mesangial cells is believed to be the key event leading to glomerulosclerosis and renal failure in diabetes (4). Consistent with the finding in clinical studies that hyperglycemia plays a key role in the development of diabetic kidney disease (17a), investigators have confirmed an important role for high extracellular glucose concentrations in stimulating mesangial cell ECM synthesis in vitro (5, 22, 25, 37, 38, 43). Facilitative diffusion of glucose into RMC has previously been reported (37). GLUT1 is the predominant facilitative glucose transporter in cultured mesangial cells (30) and is a high-affinity, low-capacitance transporter with a Km in the range of 1-7 mM for 2-deoxyglucose (30). These kinetic properties suggest that the capacity for elevations in extracellular glucose concentration to increase glucose uptake in the mesangial cell may be limited by saturation of glucose transport at or near the physiological glucose concentrations. Early increases in cellular glucose uptake by RMC in response to high extracellular glucose may occur before GLUT1 upregulation, i.e., glucose transport capacity may not be fully saturated at physiological glucose concentrations.

Glucose transporters are membrane-embedded proteins that mediate the facilitative uptake of glucose from the surrounding medium into the cell. The regulation of glucose transporters by glucose itself is poorly understood, although it appears to be tissue specific (23, 34, 36). Many cell types exposed to hyperglycemic conditions limit their increase in glucose metabolism by reducing the activity or expression of facilitative glucose transporters, mainly in tissues that are not the target of end-organ damage from diabetes (i.e., adipose and skeletal muscle) (34). Recent reports have described the upregulation of facilitative glucose transporters by pathophysiological concentrations of glucose in the retina (40, 52, 55), erythrocytes (27), and mesangial cells (29). In addition, troglitazone (54) and tumor necrosis factor-alpha (44) have been reported to stimulate glucose transport. The clinical relevance of enhanced glucose utilization and diabetic nephropathy has recently been reported by Christian et al. (14). The findings of Christian et al. (14) of increased renal glucose uptake and metabolism in human subjects with diabetes would suggest that altered glucose transport and/or metabolism is a part of the kidney response to elevations in ambient glucose in diabetes. These findings suggest that end-organs may be subject to increased risk of diabetic complications by failing to reduce glucose transporter activity or expression in the face of elevated extracellular glucose concentrations.

How increased glucose utilization activates AR, PKCalpha , and the native GLUT1 genes is not known, although preliminary data suggest that glucose increased transcription of the GLUT1 gene through putative GlRE (32). High extracellular glucose was not necessary to activate AR and increase sorbitol content in the RMCs cells. Pathophysiological levels of glucose increased AR mRNA abundance, protein activity, and sorbitol production in both the MCGT1 and MCLacZ cell. AR has classically been described as an osmoprotective gene protecting the renal medullary cells from high external osmolality (24, 35), but high extracellular glucose is not necessary to activate AR in the RMCs. Standard growth media containing 12 mM mannitol as an osmotic control had no appreciable effects on the expression of AR, PKCalpha , or GLUT1 mRNAs in the MCGT1 or MCLacZ cells (data not shown). These findings are similar to glucose-specific, nonosmotic activation of AR in the human pancreatic duct epithelial cell line CAPAN-1, as reported by our laboratory (11). CAPAN-1 human pancreatic duct cells also demonstrate an AR inhibitor reversible 29% decrease in Na-K-ATPase and a glucose-dependent increase in AR protein and AR activity (11).

Much of our present understanding of AR gene regulation has been derived from studies examining the highly conserved adaptive response of renal medullary cells to hyperosmolality (osmoregulation/compatible osmolyte hypothesis) (10, 12, 60). Although these discoveries have contributed greatly to our understanding of hyperosmotic regulation of AR in the renal medulla, little is known about AR gene regulation by pathophysiological levels of glucose seen in diabetes. In diabetes, hyperosmolarity would not appear to be sustained long or high enough to account for the increase in AR seen in end-organs affected by diabetes. Although the physiological role of AR outside of the renal medulla is not known, pathophysiological concentrations of glucose levels may provide the basis for the detrimental activation of AR in RMC.

Activation of AR and the metabolic consequences of AR activation were greater in the MCGT1 cell than in the MCLacZ cells and may reflect the 43-fold greater glucose utilization by the MCGT1 cells for glucose metabolism (35). It is not known whether the increased rate of glucose transport per se or increased glucose utilization is responsible for activation of the AR gene; however, previous reports of GlREs in the liver (LPK and S14 genes) indicate a requirement for glucose metabolism (49).

Central to the metabolism of glucose is the requirement for its phosphorylation to glucose-6-phosphate by glucokinase or hexokinase (23). It is not yet known whether phosphorylation is necessary for activation of AR, PKCalpha , or GLUT1. An as yet unidentified GlRE could be responsible for activation of AR, GLUT1, or PKCalpha in the RMCs. Increases in PKC induced by glucose in RMC rather that high glucose per se may signal increases in GLUT1 or AR expression. In preliminary studies (32), we have identified the mesangial cell GLUT1 gene as being glucose responsive and have identified two putative GlREs in the GLUT1 gene. GlREs have been studied mostly in the context of glucose-dependent regulation of gene expression such as the L-type pyruvate kinase gene (42), fatty acid synthetase (23), or the S14 gene as reviewed by Towle (58). There are common consensus sequence and transacting factors (CACGTG motifs) that are known to confer a glucose-specific response in mammalian tissues (49, 59). In a recent report, the pentose phosphate shunt intermediate xylulose-5-phosphate conferred glucose responsiveness in the L-type pyruvate kinase gene (18); hence, an intermediate of glucose metabolism and not glucose transport per se may confer glucose-specific responsiveness of these genes.

The metabolic consequences of increased glucose flux in MCGT1 cells have been partially characterized and include 1.9-, 2.5-, and 2.1-fold increase in the production of myo-inositol, sorbitol, and lactate content, respectively, as well as increases in glucose uptake (5-fold) and in net utilization of glucose (43-fold). The findings in this report also demonstrate a persistent increase in PKCalpha mRNA and total protein in the MCGT1 cells. These findings suggest increased transcription and or increased PKCalpha transcript stability and increased synthesis of PKCalpha protein in response to enhanced glucose transport. Increased PKCalpha could be the result of de novo synthesis of diacylglycerol (3) leading to production of jun and fos protooncogenes that form the transcription activator protein 1 complex (AP-1), which binds to the cis-acting sequences in the promoter of ECM genes (2). High glucose has been demonstrated to increase PCKalpha in RMC (16, 25, 39).

We have recently reported that glucose stimulates RMCs GLUT1 expression and uptake and IGF-I-sensitive glucose uptake (29). IGF-I has previously been reported to stimulate glucose uptake in cultured RMCs (26). RMCs increase IGF-I production (19), expression of IGF-I receptor (47), and IGF-I stimulated fibronectin accumulation in diabetic mesangial cells (57). In that report, medium containing 20 mM glucose increased GLUT1 mRNA (134%), GLUT1 protein (68%), and the Vmax for glucose uptake by 50% in RMCs (29). The enhanced glucose uptake was further enhanced by the addition of IGF-I to the growth media (29). Thus IGF-I may have an autocrine effect to increase GLUT-1 activity or translocation in RMCs exposed to elevated glucose. Posttranscriptional changes in AR protein stability or activity were not determined in the MCGT1 cell but could have accounted for the differences in AR mRNA, AR protein, and AR activity between MCGT1 and MCLacZ cells.

Although the findings of the Diabetes Control and Complications Trial (17a) have removed any doubt regarding the role of elevated blood glucose and the risk for long-term complications from diabetes, the mechanism(s) of tissue injury in diabetes is poorly understood. There is presently no unified biochemical or molecular theory that explains the development of long-term complications in all target organs affected by diabetes; however, the metabolism of glucose is central to all proposed theories. The rate of glucose entry or metabolism may be a key proximal event in the development of glucose-mediated tissue damage from diabetes. Our findings in RMCs suggest a detrimental positive feedback relationship may exist when the cell is unable to limit the rate of glucose uptake in the face of elevated ambient glucose concentrations. Increased transport and metabolism of glucose augmented AR activity and polyol metabolism, PKCalpha expression, increased ECM accumulation (31, 35), and native GLUT1 expression and glucose transport. Many of our present therapies to prevent the long-term complications of diabetes are directed at the normalization of blood glucose often at the expense of increased glucose transport into cells. New treatments aimed at limiting the rate of glucose transport into end-organs affected by diabetes may provide another means of preventing diabetic complications.


    ACKNOWLEDGEMENTS

The anti-AR antibody was a generous gift from Peter F. Kador, Phd, of the Laboratory of Ocular Therapeutics, National Eye Institute, Bethesda, MD. We thank Seth R. Hootman, Phd, for technical assistance in immunoblotting of aldose reductase and Cameron Deyarmon Henry for assistance in the preparation of the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Physician Scientist Award K11-DK-02193 and Juvenile Diabetes Foundation International Research Grant 195044 (to D. N. Henry), NIDDK Grant K08-DK-01953 (to C. W. Heilig), and a National Kidney Foundation of Michigan Grant. F. C. Brosius III is an established investigator of the American Heart Association.

Portions of this work were presented in abstract form at the 29th Annual Meeting, American Society of Nephrology, Nov. 3-6, 1996, in New Orleans, LA.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. N. Henry, 113 Giltner Hall, Dept. of Physiology, Michigan State Univ., East Lansing, MI 48824-1101 (E-mail: henry{at}psl.msu.edu).

Received 25 June 1998; accepted in final form 29 March 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akagi, Y., P. F. Kador, and J. H. Kinoshita. Immunohistochemical localization for aldose reductase in diabetic lenses. Invest. Ophthalmol. Vis. Sci. 28: 163-167, 1987[Abstract].

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3.   Ayo, S. H., R. Radnik, J. A. Garoni, D. A. Troyer, and J. I. Kreisberg. High glucose increases diacylglycerol mass and activates protein kinase C in mesangial cell cultures. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F571-F577, 1991[Abstract/Free Full Text].

4.   Ayo, S., R. Radnik, J. Garoni, W. Glass, and J. Kreisberg. High glucose causes an increase in extracellular matrix proteins in cultured mesangial cells. Am. J. Pathol. 136: 1339-1348, 1990[Abstract].

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