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# Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA; and
Division of Cardiology, Department of Medicine, University of Louisville, Louisville, Kentucky, USA
1Correspondence: Department of Human Biological Chemistry and Genetics Room 6.644, Basic Science Building, University of Texas Medical Branch Galveston, TX 77555-0647, USA. E-mail: ssrivast{at}utmb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: aldose reductase • glutathiolation • diabetes • nitric oxide and nitrosothiols
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Although due to insulin deficiency or resistance, glucose utilization is diminished in tissues that require insulin for glucose uptake, tissues in which glucose transport is not regulated by insulin face severe and sustained hyperglycemia (4
, 5)
. Because glycolytic utilization is saturated, excessive glucose in these tissues is converted to sorbitol via NADPH-dependent reduction catalyzed by aldose reductase (AR). Under normal, euglycemic conditions, sorbitol synthesis represents a minor (>3%) fate of glucose in nonrenal tissues, however, at levels encountered during diabetes, 30 to 35% of the glucose could be converted to sorbitol. This increase in the polyol pathway has been linked to several pathological changes in insulin-insensitive tissues such as those in the blood vessels, peripheral nerves, renal medulla, blood cells, and ocular lens. Although the mechanisms by which the increase in the polyol pathway contributes to hyperglycemic injury are not well understood, it has been suggested that the osmotic and/or oxidative stress imposed by sorbitol accumulation and NADPH depletion may be significant biochemical changes contributing to the observed pathological changes (6
, 7)
.
That a component of hyperglycemic injury is due to the increase in the polyol pathway activity is supported by extensive evidence showing that inhibition of AR prevents diabetic nephropathy, neuropathy, and cataractogenesis in rats (8
, 9)
. The contribution of AR to hyperglycemic injury is further supported by the observation that lens-specific overexpression of AR accelerates diabetic cataracts in mice (10)
. Nevertheless, the clinical utility of AR inhibitors in treating secondary diabetic complications remains unclear. Although some of the variable clinical outcomes may be related to inappropriate dosing and hypersensitivity of selected individuals, the limited long-term efficacy of these drugs may be due in part to post-translational changes in AR that alter ligand binding and catalysis. Our previous studies have shown that AR isolated from diabetic tissues displayed altered kinetic properties and was relatively insensitive to hydantoin inhibitors such as sorbinil compared with the enzyme from normal tissues (11)
. Similar changes in kinetic and ligand binding properties of AR were obtained upon in vitro thiol modification of the enzyme by hydrogen peroxide (H2O2) or NO, indicating that the intracellular activity of AR may be regulated by redox-sensitive reactions.
The high sensitivity of AR to oxidants such as H2O2 and NO is due to a reactive cysteine (Cys-298) present at the active site of the enzyme (12)
. We have shown that Cys-298 is readily modified by NO donors and that depending on the conditions of the reaction and the nature of the NO donor used, the enzyme is either S-thiolated or S-nitrosated (13
, 14)
. On the basis of these observations, we hypothesized that NO regulates intracellular activity of AR and consequently the flux of glucose via the polyol pathway. To test this hypothesis, we examined whether changes in NO synthesis or bioavailability affect AR activity or sorbitol synthesis in aorta from diabetic or nondiabetic animals. Our results show that NO inactivates AR and inhibits sorbitol synthesis and that this may relate to reversible S-thiolation of AR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vivo regulation of polyol pathway in normal and diabetic rat aorta
To investigate the in vivo effects of NO, diabetes was induced in 
3-month-old Sprague-Dawley rats by injecting streptozotocin (STZ; 65 mg/kg body wt). Only those rats that had blood glucose levels > 400 mg% on the 4th day of the STZ injection were used in the study (group II). Nondiabetic and diabetic rats were divided in four groups each. Groups I to IV were nondiabetic and groups V-VIII were diabetic. Groups I and V were injected with the carrier; groups II and IV with L-arginine (200 mg/kg body wt); groups III and VII with L-NAME (50 mg/kg body wt); in groups IV and VIII nitroglycerine patches that release 200 ng NO/min (calculated on the basis of manufacturer’s manual, Schwarz Pharma) were applied to the preshaved dorsal neck region and replaced every day. After 10 days of treatment, rats were killed and their aortas were removed. The aorta was homogenized in 1 mL of PBS containing 20 µL of the protease inhibitor cocktail. AR activity and sorbitol content of the homogenates were measured. Data are presented as mean ± SD and the P values were determined by unpaired Students’ t test using Microsoft Excel 2000.
Regulation of AR activity and sorbitol accumulation in aorta ex vivo
The abdominal aorta was dissected from Sprague-Dawley rats, C57/BL-6 mice, or the eNOS null mice in the C57/BL6 background (obtained from Jackson Laboratories, West Grove, PA, USA). The aorta was dissected into six 5 mm strips. Aortic strips from 6 to 8 animals were pooled and divided into groups with 6 random pieces in each group. The aortic strips were incubated in M-199 medium containing 10% FBS, 1% penicillin/streptomycin, and 2 µg/mL cycloheximide in the absence or presence of 2 mM L-arginine or 1 mM L-NAME at 37°C in a humidified CO2 incubator. After 12 h of incubation, 50 mM glucose was added to the medium and the incubation was continued for another 24 h. The samples were washed with ice-cold PBS and homogenized in 1 mL of 0.1 M phosphate (pH 7.4) containing protease inhibitor cocktail; AR activity and the sorbitol content were measured (15
, 16)
.
Cell culture and treatment
The VSMC were maintained and grown to confluency in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2. Before addition of the NO donors, the medium was replaced with Krebs-Hansliet (KH) buffer containing (in mM): NaCl, 118; KCl, 4.7; MgCl2, 1.25; CaCl2, 3.0; KH2PO4, 1.25; EDTA, 0.5; NaHCO3, 25; glucose 5, pH 7.4. Freshly prepared solutions of the nitric oxide donors (SNAP, SIN-1, GSNO, GSNO-ester, NOC-9, or NONOate) or AR inhibitors (sorbinil and tolrestat) at a final concentration of 1 mM were added to the culture medium. In some experiments, SNAP was added to the VSMC cultured in the presence of DMEM with 10% FBS. Samples were incubated at 37°C under 5% CO2 for 2 h, then 40 mM glucose was added to the incubation medium and the incubation was continued an additional 4 h. For regeneration of AR activity, the VSMC were incubated with NO donors for 2 h, followed by replacement of the media with fresh media without NO donors; the incubation was continued for an additional 6 h. The cells were harvested and lysed in 10 mM phosphate (pH 7.0) containing 20 µL of the protease inhibitor cocktail. An aliquot of the sample was removed to determine the total protein content, and AR enzyme activity and the rest of the sample was used to measure sorbitol.
Measurement of AR and sorbitol
Tissues or cells were homogenized in 1 mL of 0.1 M phosphate (pH 7.4) containing protease inhibitor cocktail. The AR activity was measured using glyceraldehyde as substrate as described previously (15
, 16)
. For sorbitol measurements, proteins in the homogenate (0.5 mL) were removed by precipitating with Ba(OH)2 and ZnSO4 (0.5 M each). The 10,000 x g supernatants were ultrafiltered using Amicon YM-10 microcon and lyophilized. The lyophilized samples were dried overnight in a vacuum desiccator over CaCl2 and derivatized by adding 0.1 mL of Deriva-Sil. One microliter of the derivatized sample was applied to a Varian 3400 gas chromatograph coupled to a hydrogen flame ionization detector. The sugars were separated on a Chrompack capillary column packed with CP Sil 24CB. The column temperature was set at 140°C and programmed to increase at a rate of 4°C/min to 170°C, then to 250°C at a rate of 50°C/min. The temperature was then held constant for an additional 3 min. The injection port was maintained at 250°C and detector temperature was set at 300°C. The amount of sorbitol in the sample was calculated using reagent sorbitol derivatized and processed using an identical protocol.
Metabolic labeling of VSMC and immunoprecipitation of AR
The medium from the flask containing confluent VSMC was removed and the cells were washed with the KH buffer. The cells were then reincubated with the KH buffer containing 2 µg/mL of cycloheximide (to inhibit protein synthesis) at 37°C in 5% CO2. After 60 min of incubation, 20 µmol/mL L-[35S]-cysteine was added to the flask and the cells were incubated an additional 5 h to label the glutathione pool. The metabolically labeled cells were incubated with SNAP for the indicated durations. To immunoprecipitate AR, cells were lysed with cold Tris-Triton buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na2O2V7, 0.2 mM PMSF, 0.5% NP-40, and 20 µL of protease inhibitor cocktail) and centrifuged at 10,000 x g for 5 min at 4°C. An aliquot of the supernatant was used to measure the protein concentration. To 500 µg of total lysate protein, 2 volumes of immunoprecipitation buffer (2% Triton X-100, 300 mM NaCl, 20 mM Tris pH 7.4, 2 mM EDTA, 2 mM EGTA, 0.4 mM Na2O2V7, 0.4 mM PMSF, 1.0% NP-40, and 20 µL of protease inhibitor cocktail) and 50 µg of affinity-purified AR antibodies were added and the samples were incubated at 4°C for 2 h. After the incubation, 100 µL of protein-A agarose beads was added and the samples were incubated overnight on a shaker at 4°C to precipitate free and bound IgG. The samples were centrifuged at 10,000 x g for 5 min and washed twice with immunoprecipitation buffer. The pellet was resuspended in 50 µL of 250 mM Tris pH 6.8 containing 4% SDS, mixed, and centrifuged at 10,000 x g for 5 min. The supernatant was used for SDS-PAGE using 10% gel. The gel was then dried and autoradiographed.
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RESULTS |
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). The dramatic 22-fold difference in the sorbitol content of the diabetic and nondiabetic aorta was correlated with a 20-fold higher AR activity in the homogenates of aorta from diabetic rats vs. nondiabetic rats. These results demonstrate that diabetes is associated with a marked up-regulation of the polyol pathway, which could be accounted for by a parallel increase in the AR activity, and that the diabetic changes in the pathway lead to a net accumulation of sorbitol in the vessel wall.
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To examine whether NO affects vascular activity of the polyol pathway, nondiabetic and diabetic rats were treated with L-arginine, a substrate of nitric oxide synthase (NOS) that, when delivered systemically, increases NO production. As shown in Table 1
, the L-arginine-treated rats accumulated 25% less sorbitol in the aorta than untreated animals. The inhibitory effects were more pronounced in diabetic rats: sorbitol content of the aorta was 80% lower than with the untreated animals. The decrease in sorbitol accumulation in diabetic and nondiabetic aorta upon L-arginine treatment was accompanied by a corresponding inhibition of AR activity. Application of the nitroglycerine patches also resulted in decreased levels of sorbitol and AR activity in the diabetic and nondiabetic aorta. However, sorbitol levels and AR activity decreased less dramatically than that observed with L-arginine (Table 1)
. Collectively, these observations indicate that NO inhibits AR and polyol accumulation in the aorta of diabetic and nondiabetic rats. To test the converse case, i.e., inhibition of NO synthesis promotes sorbitol accumulation, we examined the effects of the NOS inhibitor L-NAME. As shown in Table 1
, treatment with L-NAME led to a 1.7-fold increase in sorbitol accumulation in the nondiabetic rats and a 3-fold increase in diabetic rats. These changes were accompanied by a proportionate increase in AR activity (Table 1)
, suggesting that inhibiting NO synthesis increases sorbitol accumulation and AR activity.
Acute regulation of AR by NO
Chronic changes in AR activity and sorbitol accumulation in the aorta of nondiabetic and diabetic animals are likely to be due to multiple processes and regulatory influences. Hence, to assess whether NO could acutely affect AR activity, we examined the role of NO in regulating sorbitol accumulation in ex vivo preparations of aorta. Ex vivo changes in the polyol pathway are unlikely to be modulated by NO-induced changes in hormones and cytokines, which could influence the polyol pathway. To minimize the confounding influence of NO on protein expression, the incubation medium was supplemented with cycloheximide to inhibit protein synthesis. Under these conditions, incubation of the aortic strips with 50 mM glucose resulted in significant accumulation of sorbitol (Fig. 1
). The accumulation of sorbitol in the aortic strips of eNOS-deficient mice was, however, significantly greater than those prepared from the wild-type (C57/BL6) mice, indicating that the lack of eNOS promotes sorbitol accumulation. Addition of L-arginine to the medium completely abolished the sorbitol accumulation and inhibited AR activity in the aortic strips prepared from nondiabetic Sprague-Dawley rats or C57/BL6 mice. However, L-arginine did not inhibit either AR activity or sorbitol accumulation in the aortic strips of eNOS null mice (Fig. 1)
, indicating that the inhibitory effects of L-arginine are entirely due to its ability to stimulate NO synthesis via eNOS and that it does not directly influence AR activity or sorbitol formation. Similarly, inhibition of NOS by L-NAME led to a significant increase in the AR activity and sorbitol accumulation in the aortic strips prepared from Sprague-Dawley rats or C57/BL6 mice. However, L-NAME had no significant effect on either the AR activity or sorbitol accumulation in aorta strips prepared from eNOS null mice. Together, these data suggest that the ability of L-NAME and L-arginine to modulate the vascular activity of the polyol pathway is due entirely to their effects on eNOS and that NO generated by the endothelium is a key modulator of polyol synthesis. These data provide additional evidence supporting the observations made in situ that increased NO generation leads to an increase in AR activity and sorbitol accumulation, whereas inhibition of NO synthesis has the opposite effect. Moreover, because the ex vivo effects were observed in the absence of protein synthesis, they suggest the possibility that post-translational modification of AR may be a significant mechanism by which NO regulates the polyol pathway.
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Effect of NO donors on VSMC
To probe the post-translational mechanism by which NO regulates AR, we used cultured VSMC in which NO levels could be controlled readily in a homogeneous cell population without using NOS inhibitors or activators. For these experiments, the confluent VSMC were incubated in KH buffer with several concentrations of SNAP ranging from 0.25 to 2.0 mM for 2 h, after which the cells were harvested, lysed, and used for measuring sorbitol and AR. Incubation with SNAP led to a dose-dependent decrease in AR activity (data not shown). Incubation with 1 mM SNAP led to a progressive decline in the enzyme activity, and maximum (80%) inhibition was observed after 2 h of incubation with 1 mM SNAP (Fig. 2
). When the SNAP-containing medium was removed and cells were reincubated in SNAP-free medium, a progressive increase in the AR activity was observed and >85% of the activity was restored, indicating that the inhibition of AR by SNAP was readily reversible.
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To prevent nonspecific binding of NO to serum proteins, the experiments with SNAP were conducted in serum-free KH medium. However, removal of serum could adversely affect the viability of VSMC or initiate signaling events, which could affect the regulatory role of NO. Therefore, in one series of experiments we incubated the VSMC with SNAP in DMEM containing 10% FBS. In these experiments, AR activity was inhibited by SNAP even in the presence of the serum, although five times more SNAP (5 mM) was required to inhibit 60% of the enzyme activity in 6 h (data not shown). These observations suggest that inhibition of AR by SNAP persists in the presence of serum and is not secondary to the stress induced by serum withdrawal. To ascertain that the inhibition of AR was due to NO and not restricted to SNAP, we investigated the effects of other NO donors and tested whether scavenging NO could abolish AR inhibition. As shown in Table 2
, incubation of VSMC with KH buffer containing 1.0 mM each of SNAP, GSNO, GSNO-ester, NONOate, or NOC-9 resulted in a 60–80% decrease in the AR activity.
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To examine the cellular consequences of inhibiting AR, we measured changes in the sorbitol accumulation. The sorbitol levels of VSMC incubated in medium containing 5.5 mM glucose were low (10 nmol/mg protein). However, when the cells were incubated with 40 mM glucose for 4 h, high concentrations of sorbitol (up to 150 nmol/mg protein) were accumulated. To test whether sorbitol accumulation in these cells was due to AR, the effects of two structurally different AR inhibitors were studied. As shown in Table 2
, incubation with tolrestat or sorbinil inhibited 95 to 97% of sorbitol accumulation. These results show that the generation of sorbitol in these cells is entirely, if not exclusively, due to AR-mediated reduction of glucose. When the VSMC were incubated with the NO donors, there was a marked decrease in cellular sorbitol content compared with untreated cells incubated in the medium without the NO donors. The extent of inhibition of sorbitol accumulation was comparable to the extent of inhibition of AR activity. No inhibition of AR was observed with the non-NO-containing analogs of these compounds (data not shown), indicating that the inhibition was specifically due to the release of NO. Furthermore, inhibition of AR activity by SNAP was prevented by the NO scavenger PTIO, confirming that the inhibition of AR was due to NO released from SNAP and not to nonspecific effects of the donor itself. Thus, together these series of experiments show that NO inhibits AR in VSMC in culture and that this inhibition prevents sorbitol accumulation and is readily reversed upon removing NO.
S-Thiolation of AR
Our previous studies show that incubation of recombinant AR with GSNO leads to glutathiolation of the enzyme at Cys-298. To examine whether NO donors S-thiolate the AR protein in VSMC, these cells were preincubated with [35S] L-cysteine in the presence of the protein synthesis inhibitor cycloheximide to prevent direct incorporation of the label in the cellular proteins and generate an intracellular pool of [35S]-labeled GSH. After metabolic labeling, the cells were incubated with 1 mM SNAP; the AR protein was immunoprecipitated using anti-AR antibodies and separated by SDS-PAGE under reducing and nonreducing conditions. As shown in Fig. 3
A, the protein band corresponding to AR immunoprecipitated from SNAP-treated cells was associated with a greater intensity of radioactivity than the AR from untreated cells, and the intensity of the band increased with increasing duration of exposure. Maximal labeling of the protein was achieved in 2 h, which corresponds in time to the progressive inhibition of VSMC AR upon SNAP treatment. Replacement of the incubation solution with the culture media without SNAP resulted in a significant loss of [35S] label from AR in 6 h. Moreover, the radioactivity associated with AR was considerably diminished when the protein was separated on reducing gels containing ß-mercaptoethanol (Fig. 3B
), demonstrating that the label was incorporated in the protein via a disulfide bond. Finally, to investigate the possibility that SNAP might decrease the AR activity by suppressing protein levels of AR, equal amounts of the immunoprecipitate were loaded on SDS-PAGE and Western blot analysis was performed using anti-AR antibody. No changes in the AR protein levels (Fig. 3C
) suggest that the differences in the radioactivity associated with the AR band could not be accounted for by changes in protein expression and are specifically due to S-thiolation of AR in the SNAP-exposed cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 18)
. Several studies have demonstrated that the reduction of glucose by AR is associated with the development of secondary diabetic complications (see beginning of article); consequently, the enzyme has been extensively studied as a pharmacological target for the treatment and prevention of diabetic complications. Nonetheless, the physiological behavior of the enzyme remains obscure and cellular mechanisms that regulate its expression and activity in nondiabetic and diabetic tissues are poorly understood. In vitro studies with homogeneous enzyme preparations have shown that AR is highly sensitive to oxidants and is readily modified by thiol-active reagents, including NO donors and nitrosothiols. This modification is highly specific because, despite the presence of three solvent-exposed cysteine residues, the enzyme is invariably oxidized at Cys-298 located at the active site of the enzyme (13
, 14)
. Modification of the enzyme by NO donors has also been localized to Cys-298, which could be stoichiometrically nitrosated or S-thiolated. The specificity and avidity with which AR reacts with the NO donors suggested to us the possibility that NO may be an endogenous regulator of AR and that, by controlling AR activity, NO could regulate glucose metabolism via the polyol pathway. The findings of the present study provide multiple lines of evidence consistent with the view that NO is indeed a critical modulator of polyol pathway of glucose metabolism and that it diminishes the activity of this pathway by inhibiting AR. Our results further suggest that the loss of NO-mediated regulation activates AR and up-regulates polyol synthesis.
The regulatory role of NO as an endogenous inhibitor of AR was evident in in situ and ex vivo models as well as in cell culture studies. In situ experiments with nondiabetic and diabetic rats showed that increasing NO generation (by providing L-arginine) or availability (by nitroglycerine patches) inhibits AR and diminishes the aortic accumulation of sorbitol. These effects were more pronounced in diabetes, which by itself led to a dramatic increase in AR activity and sorbitol accumulation. These data indicate that physiological, pathological, or pharmacological states associated with increased NO generation are likely to be invariably associated with an inhibition of the polyol synthesis in vascular tissue. Conversely, decreases in NO synthesis or availability will up-regulate AR catalysis and increase the flux of glucose via the polyol pathway. This view is supported by the observation that inhibition of NOS by L-NAME led to nearly a twofold increase in AR activity and sorbitol accumulation in both diabetic and nondiabetic rats and that sorbitol formation in the aorta of the eNOS null mice was threefold higher than that in WT mice (Fig. 1)
. From these observations, we infer that NO exerts an inhibitory influence on AR activity and the polyol pathway.
The second messenger NO is a diffusible gas that regulates several physiological processes, including blood pressure, platelet aggregation, and neurotransmission (19
20
21)
. Recent studies show that NO regulates glucose and oxygen consumption in the heart (22
23)
. Our observations that NO inhibits AR and sorbitol synthesis identifies, for the first time, a new role of NO in regulating the vascular metabolism of glucose. Unlike heart and liver, the uptake of glucose by the blood vessel wall is not regulated by insulin. As a result, hyperglycemic states such as diabetes mellitus lead to an increase in glucose in the endothelium and smooth muscle cells. Consequently, long-term diabetes is associated with multiple vascular complications that are the leading cause of morbidity and mortality in diabetics (24)
. Although the mechanisms of such vascular injury are not well understood, our results showing a profound increase in AR activity and sorbitol accumulation in diabetic aorta suggest that the polyol pathway may be a significant source of hyperglycemia-induced vascular pathology. As suggested for other tissues, an increase in the polyol pathway could deplete reducing equivalents and promote accumulation of the cell-impermeable osmolyte, sorbitol in the vessel wall. Together, these changes could impose oxidative and osmotic stress (6
, 7)
. Under these conditions, the generation and availability of NO could play a decisive role in preventing these pathological changes. Because in the aorta (as in the kidney, nerves, or retina) the uptake of glucose is not regulated by insulin, NO may be the only form of local control of glucose metabolism not intrinsic to the glucose-using enzymes themselves.
Extensive investigations show that diabetes is associated with the impairment of NO-mediated vascular relaxation and a decrease in NO bioavailability, which may be a causative factor in other complications as well (24)
. Our observation that NO tightly and dynamically controls the vascular activity of the polyol pathway, a key mediator of hyperglycemic injury, indicates that the unchecked activity of this pathway may be a critical consequence of decreased NO during diabetes. Thus, restoring NO could prevent excessive glucose reduction and the associated biochemical and pathological changes. Our observation that inhibition of NO synthesis increases polyol accumulation even in nondiabetic animals raises the possibility that in insulin-insensitive tissues, decreased NO generation or availability could induce diabetes-like changes even without hyperglycemia or changes in insulin sensitivity.
Nitric oxide could regulate the polyol pathway at multiple levels. Previous studies have shown that incubation of VSMC with NO donors results in the transcriptional up-regulation of AR (25)
. However, results of the present study suggest that direct post-translational modification of AR is likely to be a significant mechanism by which NO regulates AR. Our ex vivo experiments show that increased generation of NO inhibits AR and sorbitol synthesis, whereas inhibition of NOS causes a marked increase in AR activity and sorbitol accumulation. Because in these experiments sorbitol synthesis was measured in the presence of cycloheximide, it is unlikely the changes observed could be accounted for by changes in AR expression. The observation that neither L-NAME nor L-arginine could affect sorbitol accumulation in eNOS-deficient aorta rules out any nonspecific effects of these additives and suggests that, in these assays, NO derived from the endothelium is the only significant regulator of AR activity and sorbitol synthesis.
Additional evidence consistent with NO-mediated post-translational regulation of AR is provided by the experiments with VSMC, which show that direct exposure to NO donors inhibits AR and sorbitol formation. These changes were accompanied by S-thiolation of the enzyme. Along with our previous data showing that nitrosothiols readily induce stoichiometric thiolation of AR (13
, 14)
, the present results indicate that NO inhibits the polyol pathway by S-thiolating AR. Several of the physiological effects of NO are mediated by its high affinity for heme-containing proteins such as guanylate cyclase, hemoglobin, and cytochrome c (26
, 27)
. However, NO could also regulate protein structure and function by inducing specific modification such as nitrosation, nitration, and S-thiolation (28
, 29)
. Thiol modification in particular has been suggested to be an important physiological mechanism of redox-based signaling, and S-thiolation of several enzymes such as glyceraldehyde-3-phosphate dehydrogenase has been demonstrated (30)
. Thus, our results demonstrating for the first time that NO promotes S-thiolation of AR in VSMC corroborate our previous structural and kinetic studies and provide an additional example of redox based NO signaling. On the basis of these observations, we speculate that in thiol-replete cells, NO maintains a significant fraction of AR in the inactive state by S-thiolation, whereas a decrease in NO (e.g., during diabetes) removes this repression and stimulates AR to shuttle more glucose through the polyol pathway. Because inhibition of NO prevented sorbitol accumulation even in nondiabetic aorta, it appears that basal NO generation is sufficient to maintain a significant fraction of AR in a repressed state; it is expected that this repression is even greater under conditions (e.g., shear stress or acetylcholine stimulation) when NO generation is enhanced.
In summary, the present study shows that NO regulates AR activity and consequently the vascular metabolism of glucose via the polyol pathway. Our results suggest that NO inhibits AR by reversible S-thiolation of the protein. These observations indicate a new role of NO in regulating glucose metabolism in insulin-insensitive tissues, where it could function in parallel to insulin. Our results also suggest new avenues for understanding glucose homeostasis and treating the vascular complications of diabetes.
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ACKNOWLEDGMENTS |
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Received for publication August 16, 2002. Accepted for publication November 22, 2002.
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