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Role of Aldose Reductase and Oxidative Damage in Diabetes and the Consequent Potential for Therapeutic Options

Department of Human Biological Chemistry and Genetics (S.K.S., K.V.R.), University of Texas Medical Branch, Galveston, Texas 77555; and Division of Cardiology (A.B.), Department of Medicine, University of Louisville, Louisville, Kentucky 40202

Correspondence: Address all correspondence and requests for reprints to: Satish K. Srivastava, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555. E-mail: ssrivast{at}utmb.edu

	Abstract

Top Abstract I. Introduction: Aldose... II. AR and Antioxidant... III. Redox Regulation of... IV. Regulation of Intracellular... V. AR Inhibitors and... VI. Conclusion References

 
Aldose reductase (AR) is widely expressed aldehyde-metabolizing enzyme. The reduction of glucose by the AR-catalyzed polyol pathway has been linked to the development of secondary diabetic complications. Although treatment with AR inhibitors has been shown to prevent tissue injury in animal models of diabetes, the clinical efficacy of these drugs remains to be established. Recent studies suggest that glucose may be an incidental substrate of AR, which appears to be more adept in catalyzing the reduction of a wide range of aldehydes generated from lipid peroxidation. Moreover, inhibition of the enzyme has been shown to increase inflammation-induced vascular oxidative stress and prevent myocardial protection associated with the late phase of ischemic preconditioning. On the basis of these studies, several investigators have ascribed an important antioxidant role to the enzyme. Additionally, ongoing work indicates that AR is a critical component of intracellular signaling, and inhibition of the enzyme prevents high glucose-, cytokine-, or growth factor-induced activation of protein kinase C and nuclear factor--binding protein. Thus, treatment with AR inhibitors prevents vascular smooth muscle cell growth and endothelial cell apoptosis in culture and inflammation and restenosis in vivo. Additional studies indicate that the antioxidant and signaling roles of AR are interlinked and that AR regulates protein kinase C and nuclear factor-B via redox-sensitive mechanisms. These data underscore the need for reevaluating anti-AR interventions for the treatment of diabetic complications. Potentially, the development of newer drugs that selectively inhibit ARmediated glucose metabolism and signaling, without affecting aldehyde detoxification, may be useful in preventing inflammation associated with the development of diabetic complications, particularly micro- and macrovascular diseases.

I. Introduction: Aldose Reductase and Diabetic Complications

II. AR and Antioxidant Protection

III. Redox Regulation of AR Activity

IV. Regulation of Intracellular Signaling by AR

V. AR Inhibitors and the Treatment of Secondary Diabetic Complications

VI. Conclusion

	I. Introduction: Aldose Reductase and Diabetic Complications

Top Abstract I. Introduction: Aldose... II. AR and Antioxidant... III. Redox Regulation of... IV. Regulation of Intracellular... V. AR Inhibitors and... VI. Conclusion References

 
ALDOSE REDUCTASE (AR) is a monomeric reduced nicotinamide adenine dinucleotide (NAD) phosphate (NADPH)-dependent enzyme and a member of aldo-keto reductase superfamily. The enzyme was described in 1956 by Hers (1) as a glucose-reducing activity. It was subsequently reported by van Heyningen (2) that high levels of AR activity are present in the rat lens and that during diabetic and galactosemic cataractogenesis, AR-derived polyols—sorbitol and galactitol—accumulate in the ocular lens. Building on this observation, Kinoshita et al. (3) and Varma and Kinoshita (4) demonstrated that treatment with pharmacological inhibitors of AR ameliorated cataractogenesis in diabetic rats and galactose-exposed rabbits. Based on these observations it was proposed that accumulation of sorbitol in the lens, due to AR-catalyzed reduction of glucose, causes osmotic swelling resulting in ionic imbalance and protein insolubilization leading to cataractogenesis (5, 6, 7). A similar sequence of events could also account for hyperglycemic injury associated with diabetic retinopathy, nephropathy, and neuropathy (Fig. 1).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Involvement of polyol pathway in diabetic complications. During hyperglycemia, reduction of glucose to sorbitol by AR constitutes the first and the rate-limiting step of the polyol pathway that converts glucose to fructose via sorbitol dehydrogenase (SDH). In this pathway both NADPH and NAD+ are consumed as cofactors for the enzymes AR and SDH. Osmotic stress due to accumulation of sorbitol and oxidative stress due to changes in the ratio of NADPH/NADP+ and reduced NAD (NADH)/NAD+ are the major cause of various complications of secondary diabetes. ROS, Reactive oxygen species.

In the mid-1980s we demonstrated that most of the inhibitors synthesized by pharmaceutical companies were not selective for AR but also inhibited other members of the aldo-keto reductase superfamily such as aldehyde reductase (20). In addition, we also demonstrated that increased osmotic pressure due to accumulation of polyols in galactosemic and diabetic cataractogenesis could not be the main cause of cataractogenesis (21, 22, 23). Antioxidants such as butylated hydroxytoluene and 6-hydroxy-2,5,7,8-tetramethenyl-chroman-2-carboxylic acid (Trolox) prevented diabetic cataractogenesis in rats and significantly attenuated lens opacity in galactose-fed rats even though the polyol levels in the lens were extremely high (80–90 mM), indicating that polyol accumulation per se is not sufficient for cataractogenesis and that other metabolic changes accompanying AR activation may be critical and important modulators of the cataractous effects of hyperglycemia and diabetes (Fig. 2).

View larger version (15K): [in this window] [in a new window]   FIG. 2. Oxidative stress is the main contributor to diabetic cataractogenesis. Digital image analysis was performed on lenses cultured in normal glucose (5.5 mM; NG), high glucose (50 mM; HG) without or with antioxidants on d 8. Sorbitol content was measured by using gas chromatography. The data represent mean ± SD (n = 4). **, P < 0.001; #, P = nonsignificant in HG-treated group compared with NG-treated group. BHT, Butylated hydroxytoluene.

Another reason for the inconsistent effects of AR inhibitors may relate to posttranslation modification of the enzyme. As documented extensively in our previous publications (26, 27, 28), the oxidation state of a single cysteine residue located at the active site of the enzyme regulates both substrate and inhibitor binding and thus, given the extensive oxidative changes in diabetes, it is likely that in diabetic tissues AR is insensitive to inhibitors due to oxidative modification. In support of this view, we have demonstrated that AR in human erythrocytes exists in two forms, activated and unactivated, and in hyperglycemia total activity of AR increases (29). Multiple forms of AR have also been reported by other investigators (30, 31), and our in vitro data suggest that oxidants can induce a variety of changes in the catalytic activity of the enzyme, although which of these forms occur in vivo and how they are interconverted remains unclear. Nevertheless, the view that the hyperglycemia changes the sensitivity of AR to inhibition is further supported by the observation that prolonged culture of endothelial cells in high glucose progressively decreases the efficacy of sorbinil in preventing sorbitol accumulation (32).

In contrast to inhibitor data, newer molecular approaches have provided more unambiguous evidence for the involvement of AR in diabetic complications. In these studies transgenic overexpression of AR gene selectively in the lens was found to accelerate diabetic and galactosemic cataract formation in mice (33). Additionally, ubiquitous overexpression of the AR gene increased the rate of neuropathic changes in diabetic animals (34, 35, 36). Collectively, these data argue strongly that AR is an important component of high glucose-induced metabolic changes that underlie the development of secondary diabetic complications. Nevertheless, despite this evidence, the exact mechanism by which AR contributes to the development of diabetic complications remains unclear. Persistent increase in extracellular glucose levels induces pleiotrophic changes in metabolism that elicit polygenic responses (Fig. 3). Extensive changes in the activation of protein kinases and the accumulation of advanced glycation end products have been reported (37, 38, 39), although their relationships with modulatory influences on AR remain unclear. In principle, AR could contribute to both protein kinase activation and advanced glycation end product accumulation. In addition, AR could catalyze the formation of potent protein glycating agents and induce oxidative stress.

View larger version (39K): [in this window] [in a new window]   FIG. 3. Pathways of hyperglycemia-induced oxidative stress. Increased superoxide production in the mitochondria during hyperglycemia causes increased oxidative stress. During hyperglycemia the ratio NADPH/NAD+ decreases due to excessive use of NADPH for the reduction of glucose to sorbitol. Consequently, the NADPH availability to maintain glutathione in the reduced form catalyzed by glutathione reductase decreases, which causes oxidative stress. Increase in advance glycation end products (AGEs) formation and binding of AGE to its cognate receptor (RAGE) also increases oxidative stress. Increased DAG formation in hyperglycemia activates PKC and subsequently induces oxidative stress via a PKC-dependent activation of NAD(P)H oxidase.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Pathways of increased DAG in hyperglycemia. Increased stress conditions because of autooxidation of glucose, osmotic stress, oxidative stress, and hypoxia during polyol pathway hyperactivity can contribute to increase in DAG levels through a cascade of signals that activate phospholipase C (PLC) or directly through glycolysis. AGE, Advance glycation end product; ROS, reactive oxygen species; SDH, sorbitol dehydrogenase.

	II. AR and Antioxidant Protection

Top Abstract I. Introduction: Aldose... II. AR and Antioxidant... III. Redox Regulation of... IV. Regulation of Intracellular... V. AR Inhibitors and... VI. Conclusion References

View larger version (47K): [in this window] [in a new window]   FIG. 5. Reduction of lipid aldehydes by aldose reductase. The bold numbers in parentheses represent reference numbers.

Based on our substrate specificity studies showing high-affinity reduction of glutathione conjugates by AR (66, 67), we reasoned that the active site of the enzyme must conform to a specific glutathione-binding domain. Consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site, we found that alterations in the structure of glutathione diminished the catalytic efficiency for the reduction of glutathione-aldehyde conjugates and that nonaldehydic conjugates of glutathione or glutathione analogs displayed active site inhibition (67). Molecular dynamics calculations suggest that the conjugates adopt a specific low-energy configuration at the active site (Fig. 6). Mutations of the active site residues identified by these calculations selectively decreased catalysis of the glutathione-aldehyde conjugates, without affecting reduction of the free aldehyde. The high specificity and selectivity with which the enzyme catalyzes the reduction of glutathione conjugates suggest that such conjugates may be in vivo substrates of the enzyme. Indeed, our metabolic studies with 4-hydroxynonenal (HNE) show AR-dependent reduction of its glutathione adduct in heart, smooth muscle, and erythrocytes (78, 79, 80, 81).

View larger version (30K): [in this window] [in a new window]   FIG. 6. Molecular modeling showing two possible orientations of glutathione-propanal bound to AR. Orientations 1 and 2 are shown in panels A and B, respectively. Carbon, nitrogen, sulfur, and oxygen atoms are colored gray, blue, yellow, and red, respectively. Potential hydrogen bonds are indicated with dashed lines. [Reproduced with permission from B. L. Dixit et al.: J Biol Chem 275:21587–21595, 2000 (67 ).]

Consistent with its role as a detoxification enzyme, AR has been found to be regulated by aldehydes generated from lipid peroxidation (64, 70, 82), thiol-reducing agents (83, 84, 85, 86, 87, 88), metal ions (89, 90, 91), and NO (92, 93, 94, 95, 96, 97). Tissue levels of AR are also increased under conditions of high oxidative stress such as iron overload (98), alcoholic liver disease (99), heart failure (100), and vascular inflammation (53, 101). Moreover, AR is also specifically up-regulated during vasculitis, specifically in the areas of high HNE formation, and inhibition of AR increases the concentration of free HNE and protein-HNE adducts, accompanied by a 3-fold increase in the number of apoptotic cells (102). Our studies show that AR is also up-regulated in the heart by brief episodes of ischemia (103). This increase in AR was prevented by inhibitors of PKC and NO synthase, indicating that the up-regulation of AR is regulated by cardioprotective signaling associated with the late phase of ischemic preconditioning. Furthermore, a posttranslational activation of AR during myocardial ischemia has been reported (104). The functional significance of the increase in AR in the ischemic heart is underscored by the observation that inhibition of AR abolishes the cardioprotective effects of late preconditioning (105, 106, 107). Collectively, these data provide support to the view that AR is a component of an antioxidant defense mechanism, which protects tissues from the harmful effect of lipid peroxidation products such as HNE that accumulate during conditions of oxidative stress such as ischemia and inflammation.

Although HNE and related aldehydes are metabolized by several different pathways, metabolism via AR could represent a true detoxification mechanism. In most cells, HNE and related aldehydes readily form glutathione conjugates; however, the formation of glutathione-HNE itself may not be sufficient for detoxification. The glutathione-aldehyde conjugates are toxic. They induce DNA damage and stimulate radical formation (108, 109, 110). Therefore, reduction of the glutathione-aldehyde conjugates by AR may be necessary to substantially annul the reactivity of the conjugate and to decrease free radical generation. Nonetheless, the mechanisms by which AR-dependent aldehyde metabolism regulates the development of diabetic complications remain unclear, and the contribution of AR to cell growth and antioxidant defense requires further elucidation. In particular, it remains to be clarified how AR expression and activity are regulated and which components of intracellular signaling are regulated by AR.

	III. Redox Regulation of AR Activity

Top Abstract I. Introduction: Aldose... II. AR and Antioxidant... III. Redox Regulation of... IV. Regulation of Intracellular... V. AR Inhibitors and... VI. Conclusion References

 
As discussed above, a component of the hyperglycemic injury is due to an increased flux of glucose through the polyol pathway because the inhibition of AR prevents some of the diabetic complications. Although inefficacy of AR inhibitors could be due to their nonspecificity and hypersensitivity of selected individuals, the limited long-term efficacy of the AR inhibitors could be attributed to the posttranslational modification in AR that alters ligand binding and catalysis. In our earlier studies we found that AR isolated from individuals with different levels of hyperglycemia displayed altered kinetic properties and was less sensitive to hydantoin inhibitors such as sorbinil compared with the enzyme isolated from the tissues of normoglycemic subjects (29). Similar changes were observed at various stages of purification of AR in the absence of a reducing reagent such as dithiothreitol or ß-mercaptoethanol from the tissues of normal subjects or from recombinant bacteria overexpressing AR (30, 31). Both the Michaelis-Menten constant (K_m) and inhibition constant (K_i) values of the enzyme significantly increased upon its in vitro thiol modification by HNE (64, 82), nitrosoglutathione (GSNO) (92), and oxidized glutathione (GSSG) (83).

The high sensitivity of AR to oxidants such as H₂O₂ and NO was attributed to a highly reactive cysteine (Cys-298) residue present at the active site of the enzyme (88). Oxidants such as H₂O₂ cause enzyme inactivation. Also glutathiolation of the Cys-298 results in a significant inactivation of the enzyme (83, 88, 92). However, depending on the conditions of the reaction and the nature of the NO donor used, AR is either S-thiolated (inactivated enzyme) or S-nitrosated (activated enzyme). On the basis of these observations, we hypothesized that NO regulates the intracellular activity of AR and consequently the flux of glucose via the polyol pathway (92, 93, 94, 95, 96, 97, 98). Because, in hyperglycemia, NO synthesis is significantly less compared with normoglycemic subjects, it has been reasoned that the AR activity may be up-regulated in diabetic tissues (111, 112).

Cardiovascular complications are one of the major causes of mortality in patients with prolonged diabetes (113). The role of AR in cardiovascular complications, especially in atherosclerosis (100), restenosis subsequent to balloon injury (53, 57), and cardiac preconditioning (103, 104, 105, 106), has been extensively investigated. Vascular endothelial cells are the main source of NO for vascular smooth muscle cells because in the vasculature, NO synthase is present mainly in the endothelium (114). The NO secreted by endothelial cells could readily form GSNO with glutathione that is abundant in vascular smooth muscle cells (VSMC). The GSNO formed could, in turn, readily S-glutathiolate AR at Cys-298 (92). Although GSSG (50–100 µM) also S-glutathiolates AR, it is unlikely that the physiological source of S-glutathiolation of AR (or other proteins) could be GSSG because the concentration of GSSG in cells rarely exceeds 20–25 µM. Because S-glutathiolation inactivates AR, under normoglycemic conditions, it appears likely that a significant fraction of AR in vascular tissues is present in an inactive form, whereas in hyperglycemia a decrease in NADPH/NADP⁺ ratio and other factors would decrease NO and the AR would be in the active form. Indeed, we have demonstrated that, in diabetic rat aorta, both AR activity and sorbitol levels are more than 20-fold higher than nondiabetic aorta (95). Daily ip injections of NO synthase inhibitor, N^G-nitro-L-argininemethyl ester or application of nitroglycerin patch significantly increased the activity of aorta AR as well as sorbitol formation in both nondiabetic and streptozotocin-diabetic rats. On the other hand, daily injections of NO synthase substrate, L-arginine, significantly decreased AR activity and sorbitol content in aortas of both diabetic and nondiabetic rats. Similar results were obtained when aortic rings from normal and diabetic mice were incubated with L-arginine and N^G-nitro-L-argininemethyl ester, whereas, both NO synthase substrate and inhibitor had no effect on AR activity or sorbitol content of aorta from endothelial NO synthase-null mice (97). Interestingly, it was observed that S-glutathiolation of AR in VSMC by NO donors such as GSNO, which inactivates the enzyme, is reversible. Thus, the changes in glucose levels that alter the levels of NO would change the AR activity, sorbitol levels, and related effects in diabetic subjects. Hence, in addition to glycemic control, NO donors or drugs that increase NO levels could represent one treatment modality for the prevention or treatment of diabetic complications. Because hyperglycemia causes increased generation of reactive oxygen species by autooxidation of glucose and other metabolic pathways, antioxidants would also have beneficial effects. Diabetic complications such as cataractogenesis, retinopathy, and cardiovascular effects have been shown to be ameliorated by antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, Trolox, ascorbate, vitamin E, N-acetyl cysteine, pyruvate, etc. (115, 116, 117, 118, 119, 120).

	IV. Regulation of Intracellular Signaling by AR

Top Abstract I. Introduction: Aldose... II. AR and Antioxidant... III. Redox Regulation of... IV. Regulation of Intracellular... V. AR Inhibitors and... VI. Conclusion References

 
Diabetes is a major risk factor for the development of cardiovascular disease. It is associated with a 2- to 4-fold higher risk of cardiovascular disease, and it accelerates the progression and increases the severity of atherosclerotic lesion formation in peripheral, coronary, and cerebral arteries (121, 122, 123, 124, 125). Moreover, diabetics have a higher propensity for restenosis after percutaneous transluminal coronary angioplasty (126). Even though coronary stenting significantly reduces restenosis, diabetes remains a powerful predictor of in-stent restenosis (127). Processes that lead to an increase in smooth muscle cell growth in diabetic as well as nondiabetic restenotic vessels have not been identified but, given the observation that high levels of protein-HNE adducts are associated with proliferative vascular lesion, it appears that products of lipid peroxidation, such as HNE, play a significant role in modulating the growth of vascular lesion. This is supported by our observation that treatment with low concentrations (<2 µM) of HNE increases VSMC growth in culture, although at higher concentrations HNE induced apoptotic cell death (53). To test the role of HNE in vascular proliferation, we determined how changes in its metabolism via AR would affect its mitogenic activity. Surprisingly, we found that inhibition of AR prevented VSMC growth in culture (53). Serum-starved VSMC cultured in 1 µM HNE showed increased proliferation compared with cells cultured without HNE, as determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and cell count, whereas under similar conditions, HNE caused apoptosis in vascular endothelial cells and lens epithelial cells (Fig. 7). Both proliferation and apoptosis were attenuated by inhibiting AR using two structurally different AR inhibitors, sorbinil and tolrestat, and also by using antisense AR or small interfering RNA. In addition to HNE-stimulated growth, smooth muscle cells growing in response to serum FGF, TNF, or high glucose were also sensitive to AR inhibitors (128). Moreover, inhibition of AR also prevented smooth muscle cell growth in vivo: we balloon-injured normal and diabetic rat carotid artery and followed restenosis by quantifying the neointima formation (101). In both normal and diabetic rats, AR inhibitor significantly (45%) prevented neointimal hyperplasia (Fig 8). Interestingly, the neointimal hyperplasia was associated with increased nuclear factor--binding protein (NF-B) activation (128). This was significantly prevented by sorbinil, suggesting that AR is required for signaling pathways responsible for NF-B activation and for the proliferation of smooth muscle cells in vascular lesions. Together, these observations suggest that AR is essential for smooth muscle cell growth induced by several mitogenic pathways and that inhibition of AR interrupts their growth in culture and in vivo (53, 57, 128).

View larger version (20K): [in this window] [in a new window]   FIG. 7. Inhibition of AR attenuates proliferation of VSMC and apoptosis of human umbilical vein endothelial cells (HUVEC) and human lens epithelial cells (HLEC). Growth-arrested cells were pretreated with or without AR inhibitors, sorbinil and tolrestat (10 µM) for 24 h followed by stimulation with TNF (2 nM) for an additional 24 h. Cell viability was determined by measuring the incorporation of [3H]thymidine (10 µCi/ml), added 6 h before the end of the experiment. The extent of cell proliferation or apoptosis is expressed as percentage change in cell viability. **, P < 0.001; *, P < 0.01; and #, P < 0.001 in high glucose (HG) + AR inhibitor group compared with HG-treated group alone.

View larger version (62K): [in this window] [in a new window]   FIG. 8. AR inhibitor attenuates NF-B activation in balloon-injured normal and diabetic rat arteries. Sections of balloon-injured arteries were obtained from nondiabetic and diabetic rats 10 and 21 d after balloon injury and were stained with anti-p65 antibodies. Immunoreactivity is evident as a dark brown stain, whereas the nonreactive areas display only the background color. The bar graph shows mean ± SEM values of the percent of neointima stained by the anti-p65 antibody. *, P < 0.05 in tolrestat vs. control; +, P < 0.05 in diabetic vs. control; and #, P < 0.05 diabetic + tolrestat vs. diabetic without tolrestat. [Reprinted with permission from K. V. Ramana et al.: Diabetes 53:2910–2920, 2004 (101 ). © 2004 American Diabetes Association.]

View larger version (49K): [in this window] [in a new window]   FIG. 9. Inhibition of AR attenuates TNF-induced activation of NF-B in VSMC and human umbilical vein endothelial cells (HUVEC). Quiescent VSMC (A) and HUVEC (B) were preincubated without or with the indicated concentrations of sorbinil for 24 h and then stimulated with 0.1 nM TNF for 1 h. The nuclear extracts were prepared, and NF-B activity was measured by EMSA. [A, Reproduced with permission from K. V. Ramana et al.: J Biol Chem 277:32063–32070, 2002 (128 ); and B, reproduced with permission from K. V. Ramana et al.: FASEB J 18:1209–1218, 2004 (134 ).]

View larger version (23K): [in this window] [in a new window]   FIG. 10. Ablation of AR attenuates cytokines, growth factors, high glucose, HNE but not phorbol 12-myristate 13-acetate (PMA)-induced activation of PKC in VSMC. The VSMC were transiently transfected with AR antisense or scrambled control oligonucleotides. Subsequently, the cells were stimulated with TNF (0.1 nM), basic FGF (bFGF) (5 ng/ml), platelet-derived growth factor (PDGF)-AB (5 ng/ml), angiotensin II (Ang-II; 2 µM), HNE (1 µM), high glucose (HG, 50 mM), or PMA (10 nM) for 4 h. The membrane-bound PKC activity was determined by using the Promega Signa TECT PKC assay system (Promega Corp., Madison, WI). Bars represent mean ± SE (n = 4). *, P < 0.001 in AR antisense group compared with control group. [A portion of the data was reproduced with permission from K. V. Ramana et al.: J Biol Chem 277:32063–32070, 2002 (128 ).]

View larger version (15K): [in this window] [in a new window]   FIG. 11. Inhibition of AR attenuates TNF-induced monocyte adhesion and cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in human umbilical vein endothelial cells (HUVEC). Quiescent HUVEC were preincubated without or with 10 µM sorbinil/tolrestat for 24 h, and then stimulated with 2 nM TNF for 24 h. After 24 h of incubation, THP-1 (monocytes) cells were added, and the incubation continued for another 12 h. Cell adhesion assays were performed using light microscope. To determine cell surface expression of adhesion molecules after stimulation with TNF, the cells were fixed in 4% formaldehyde, and the expression of ICAM-1 and VCAM-1 was measured by ELISA using anti-VCAM-1 and anti-ICAM-1 antibodies, respectively. Bars represent mean ± SE (n = 4). ***, P < 0.001 in TNF- + AR inhibitors-treated group compared with TNF-treated group. [Reproduced with permission from K. V. Ramana et al.: FASEB J 18:1209–1218, 2004 (134 ).]

	V. AR Inhibitors and the Treatment of Secondary Diabetic Complications

Top Abstract I. Introduction: Aldose... II. AR and Antioxidant... III. Redox Regulation of... IV. Regulation of Intracellular... V. AR Inhibitors and... VI. Conclusion References

 
Despite the initial promise, outcomes of clinical trials with AR inhibitors have been disappointing. Most studies reveal only modest improvement with multiple side effects. However, despite such negative data, it would be unfortunate if anti-AR therapy were to be relegated to a historical footnote. Clinical trials with AR inhibitors were designed and conducted with only limited information about the enzyme and with little or no understanding of its physiological role in processes other than the reduction of glucose. Moreover, most clinical trials were designed to examine only a limited set of physiological and pathological end points and were mostly focused on diabetic neuropathy. However, subsequent to initial clinical trials, much has been learned about the enzyme and its role in glucose metabolism, detoxification, inflammation, and growth that will be critical in redesigning clinical trials with a larger number of pathological indices and end points.

Given the extensive data showing that by catalyzing the reduction of lipid-derived aldehydes and their glutathione conjugates (62, 66, 67), AR protects against oxidative stress (101, 128), it may be beneficial to try a combination therapy with AR inhibitor and antioxidants. Because long-term diabetes is associated with increased oxidative stress, the beneficial effects of AR inhibitors may be diminished by a concurrent accumulation of lipid peroxidation products. Therefore, to prevent this, it may be necessary to concurrently administer antioxidants that prevent lipid peroxidation or enhance the expression of antioxidant enzymes that can compensate for the lack of AR. Treatment with antioxidants may also keep the enzyme in the reduced form and thus prevent drug resistance that develops due to AR modification under conditions of chronic hyperglycemia. Additionally, treatment with AR inhibitors could be restricted to a specific stage of disease development. Thus, for instance, inhibition of AR may be more beneficial during early stages of diabetes, when oxidative stress is low, than during later stages of the disease when, due to increased oxidative stress and oxidative modification of the enzyme, it may be difficult to inhibit AR or to accrue significant benefits from anti-AR interventions without further increasing oxidative stress.

Although changes in cellular metabolism and tissue injury have been monitored in both animal studies and clinical trials, changes in inflammation and inflammatory markers have not been carefully examined. In view of recent studies showing that inhibition of AR prevents multiple inflammatory pathways (Refs. 101 , 128 , and 134 ; Fig. 12), it may be necessary to examine how AR inhibitors affect systemic and local inflammation during diabetes. This appears to be particularly critical because chronic inflammation has emerged to be one of the critical features of diabetic complications, particularly cardiovascular disease. Moreover, based on data showing that inhibition of AR prevents restenosis, vascular smooth muscle growth, and endothelial cell apoptosis (101, 134, 135, 136), it may be possible to design animal and clinical studies to test the efficacy of anti-AR therapy against micro- and macrovascular complications of diabetes, which appear to be the most serious and prevalent outcomes of prolonged diabetes. Because protection against vascular changes was accompanied by an inhibition of cell signaling involved in inflammation, it appears likely that AR inhibitors may be effective against diseases other than diabetes, particularly those that are associated with high levels of cytokine generation and inflammation such as rheumatoid arthritis and sepsis. Acute intervention with AR inhibitors during sepsis appears to be particularly attractive because short-term treatment will promote recovery by inhibiting both cytokine signaling and production, without the risk of long-term treatment with AR inhibitors that may chronically increase oxidative stress.

View larger version (53K): [in this window] [in a new window]   FIG. 12. Schematic representation of AR involvement in cell signaling. Cytokines, growth factors, and hyperglycemia generate ROS and cause peroxidation of lipids resulting in the generation of toxic lipid aldehydes such as HNE. AR efficiently reduces HNE and its conjugate with glutathione (GSH) to 1–4-dihydroxy-2-nonene (DHN) and GS-DHN, respectively. The reduced products of aldehydes may be involved in the cytotoxic signaling leading to cell death or growth via activation of phospholipase C (PLC)/PKC/NF-B pathway. Paracrine effects of cytokines such as TNF amplify hyperglycemia and other signals as shown in the pathway.

	VI. Conclusion

Top Abstract I. Introduction: Aldose... II. AR and Antioxidant... III. Redox Regulation of... IV. Regulation of Intracellular... V. AR Inhibitors and... VI. Conclusion References

 
Due to its ability to reduce a wide variety of aldehydes, ranging from membrane phospholipids to glucose, AR plays a complex role in cellular metabolism and signaling. Although identified initially as a glucose-reducing enzyme, the enzyme is now believed to be an important component of antioxidant defense involved in the removal and detoxification of reactive aldehydes generated by lipid peroxidation. Inhibition of AR has been shown to prevent the development of diabetic complications in animal models; however, a critical evaluation of the clinical efficacy of AR inhibitors awaits a clearer understanding of the role of AR in regulating inflammation and cell growth. More selective and effective inhibitors are needed to specifically inhibit the cytotoxic role of AR in cell signaling without affecting its detoxification role. Such inhibitors are likely to be more effective in treating secondary diabetic complications by preventing inflammation due to chronic hyperglycemia.

	Footnotes

 
This work was supported, in part, by NIH Grants DK36118, EY01677, HL55477, and HL59378.

First Published Online April 6, 2005

Abbreviations: AR, Aldose reductase; DAG, diaceylglycerol; DHN, 1,4-dihydroxy-2-nonene; FGF, fibroblast growth factor; GSNO, nitrosoglutathione; GSSG, oxidized glutathione; HNE, 4-hydroxynonenal; NAD, nicotinamide adenine dinucleotide; NADPH, reduced NAD phosphate; NF-B, nuclear factor--binding protein; NO, nitric oxide; PKC, protein kinase C; PLC, phospholiase C; VSMC, vascular smooth muscle cells.

	References

Top Abstract I. Introduction: Aldose... II. AR and Antioxidant... III. Redox Regulation of... IV. Regulation of Intracellular... V. AR Inhibitors and... VI. Conclusion References

 

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