Contribution of Polyol Pathway to Diabetes-Induced Oxidative Stress
Stephen S.M. Chung*,,
Eric C.M. Ho*,
Karen S.L. Lam and
Sookja K. Chung*,
*Institute of Molecular Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, and Department of Medicine, The University of Hong Kong, Hong Long, China.
Correspondence to Dr. Stephen S.M. Chung, 8/F Kadoorie Biological Sciences Building, Institute of Molecular Biology, The University of Hong Kong, Pokfulam, Hong Kong, PR China. Phone: 852-22990782; Fax: 852-28171006;
ABSTRACT. Diabetes causes increased oxidative stress, whichis thought to play an important role in the pathogenesis ofvarious diabetic complications. However, the source of the hyperglycemia-inducedoxidative stress is not clear. It was found that the polyolpathway is the major contributor to oxidative stress in thelenses and nerves of diabetic mice. The first enzyme in thepathway, aldose reductase (AR), reduces glucose to sorbitol,which is then converted to fructose by sorbitol dehydrogenase(SDH). Transgenic mice that overexpress AR specifically in theirlenses showed a significant increase in oxidative stress whenthey became hyperglycemic, as indicated by a decrease in GSHand an increase in malondialdehyde in their lenses. Introducingan SDH-deficient mutation into these transgenic mice significantlynormalized the GSH and malondialdehyde levels. These resultsindicate that both enzymes of the polyol pathway contributedto hyperglycemia-induced oxidative stress in the lens. In thewild-type mice, diabetes caused a significant decrease in GSHin their sciatic nerves, indicative of oxidative stress. Inthe AR null mutant mice, diabetes did not lead to any decreasein the nerve GSH level. These results indicate that similarto the situation in the lens, AR is also the major contributorto hyperglycemia-induced oxidative stress in the nerve. Althoughincreased flux of glucose through the polyol pathway leads todiabetic lesions in both the lenses and nerve, the mechanismsmay be different. AR-induced osmotic stress seems to be thecause of diabetic cataract, whereas AR-induced oxidative stressis probably the cause of neuronal dysfunction. E-mail: smchung@hkucc.hku.hk
Diabetes causes increased oxidative stress in various tissuesas evidenced by increased levels of oxidized DNA, proteins,and lipids. Besides damaging the functions of these molecules,oxidative stress also triggers a series of cellular responses,including the activation of protein kinase C (PKC) (1,2), transcriptionfactor NF-B (3), and JNK stress-associated kinases (4), andso forth. Inappropriate activation of these important regulatorymolecules would have deleterious effects on cellular functions,and it is thought to contribute to the pathogenesis of variousdiabetic complications (5). However, it is not clear how hyperglycemialeads to increased oxidative stress. It is most likely the combinedeffects of increased levels of reactive oxygen species (ROS)and decreased capacity of the cellular antioxidant defense system.Glucose auto-oxidation (6), nonenzymatic glycation (7), andthe interaction between glycated products and their receptors(8), overproduction of ROS by mitochondria (9), and the polyolpathway (10,11) all are potential sources of hyperglycemia-inducedoxidative stress. This report focuses on the contribution ofthe polyol pathway to oxidative stress.
The polyol pathway consists of two enzymes. The first enzyme,aldose reductase (AR), reduces glucose to sorbitol with theaid of its co-factor NADPH, and the second enzyme, sorbitoldehydrogenase (SDH), with its co-factor NAD+, converts sorbitolto fructose. In animal models, treatment with AR inhibitors(ARI) was shown to be effective in preventing the developmentof various diabetic complications, including cataract, neuropathy,and nephropathy (12). It was thought that osmotic stress, fromthe accumulation of sorbitol, leads to diabetic lesions (13).Although this model may be applicable to the lens, in othertissues, such as sciatic nerve, the level of sorbitol does notcorrespond to the severity of neural dysfunction (14), suggestingthat other mechanisms may be more important in contributingto diabetic lesions. Treatment of diabetic rats with an ARIattenuated the reduction of GSH in their lenses, suggestingthat AR activity causes oxidative stress (15). However, theARI may have free radical scavenging function; therefore, thenormalization of GSH may not be due to the inhibition of AR(16). Here, we report the use of a genetic approach to demonstratethat both AR and SDH contribute to diabetes-induced oxidativestress.
Polyol Pathway and Diabetes-Induced Oxidative Stress in the Lens
Mice have low levels of AR in their lenses, and they are resistantto develop diabetic cataract. To determine the role of AR inthe pathogenesis of cataract, we developed transgenic mice thatoverexpress the human AR cDNA specifically in their lenses.Expression of the AR transgene was found only in the lens andno other tissues. Under normal rearing condition, no morphologicabnormality was detected in the lenses of the transgenic mice,indicating that overexpression of AR per se does not have anydeleterious effect on the lens. When induced to become diabeticby streptozotocin injection, the transgenic mice developed cataractat a rate proportional to the level of AR expression in theirlenses, indicating that AR is the key enzyme in the pathogenesisof diabetic cataract (17).
These lens-specific AR transgenic mice were used to determinewhether the polyol pathway activity contributes to diabetes-inducedoxidative stress (18). When the wild-type mice were inducedto become diabetic, their lenses showed no sign of experiencingoxidative stress. However, the lenses of diabetic transgenicmice had significant decrease in GSH level and significant increasein the level of malondialdehyde (MDA), indicative of oxidativestress (Figure 1). These results indicate that AR is the majorcontributor to diabetes-induced oxidative stress in the lens.Introducing a copy of the SDH-deficient mutation into the ARtransgenic mice partially normalized the GSH and MDA levelsin the diabetic transgenic mice, indicating that SDH also contributesto oxidative stress (Figure 1).
Figure 1. Polyol pathway–induced oxidative stress in diabetic lens. GSH (A) and MDA (B) of wild-type, AR (heterozygous CAR648 AR transgenic), and AR/SDH (heterozygous CAR648 AR transgenic and heterozygous SDH-deficient double mutant) mice under normal and diabetic conditions. The bars indicate mean ± SD. The P values were calculated by t test.
Polyol Pathway and Diabetes-Induced Oxidative Stress in the Nerve
Wild-type mice are susceptible to develop diabetic neuropathyas indicated by reduced nerve conduction velocity (NCV) andsigns of structural abnormality of the nervous tissues (19).To determine the role of polyol pathway in the pathogenesisof this disease, we developed AR gene knockout mice (20). Thegrowth rate and reproductive capacity of these mice were similarto that of the wild-type mice. The only observable abnormalityin the AR-deficient mice is that they drink and urinate morethan the wild-type mice, indicating a mild impairment in theirurine concentrating ability. However, this does not affect thelevels of various electrolytes in their serum. When these micewere induced to become diabetic, they showed no reduction intheir NCV, indicating that AR deficiency confers to these miceresistance to develop diabetic neuropathy (Figure 2). Whereasthe diabetic wild-type mice showed significant reduction inthe GSH level in their sciatic nerve, diabetic AR null miceshowed no change in the GSH level, indicating that the polyolpathway is the major source of diabetes-induced oxidative stressin this tissue.
Figure 2. AR in diabetic neuropathy. NCV (A) and GSH (B) levels of wild-type and AR-/- (homozygous AR null mutant) mice under normal and diabetic conditions. The bars indicate mean ± SD. The P values were calculated by one-way ANOVA.
We have shown that the polyol pathway is the major source ofdiabetes-induced oxidative stress in lens and the nerve. Thereare three potential mechanisms for the polyol pathway to contributeto oxidative stress (Figure 3). (1) AR activity depletes itsco-factor NADPH, which is also required for glutathione reductaseto regenerate GSH. Under hyperglycemic condition, as much as30% of the glucose is channeled into the polyol pathway (10),causing a substantial depletion of NADPH and consequently asignificant decrease in the GSH level. Thus, during hyperglycemia,AR activity diminishes the cellular antioxidant capacity. (2)Oxidation of sorbitol to fructose by SDH causes oxidative stressbecause its co-factor NAD+ is converted to NADH in the process,and NADH is the substrate for NADH oxidase to generate ROS (21).(3) The polyol pathway converts glucose to fructose. Becausefructose and its metabolites fructose-3-phosphate and 3-deoxyglucosoneare more potent nonenzymatic glycation agents than glucose,the flux of glucose through the polyol pathway would increaseadvance glycation end products (AGE) formation. AGE, as wellas binding of AGE to their receptors, are known to cause oxidativestress.
Figure 3. Polyol pathway–induced oxidative stress. AR competes with glutathione reductase (GR) for their co-factor NADPH, leading to a decrease in GSH. Increased NADH causes NADH oxidase (NOx) to produce ROS. Fructose-3-phosphate (F-3-P) and 3-deoxyglucosone (3-DG), metabolites of fructose, increase AGE formation. AGE and binding of AGE to receptor of AGE (RAGE) increase oxidative stress.
Although the polyol pathway causes oxidative stress in boththe lens and the nerve, its role in the development of diabeticlesion in these two tissues seemed to be different. Osmoticstress, from the accumulation of sorbitol, is a more importantfactor for the development of diabetic cataract. This was demonstratedby the fact that administration of vitamin E and vitamin C,even though significantly normalized GSH and MDA levels in thediabetic lens, could not prevent the development of cataract.It only delayed the onset of cataract for a couple of days (18).Furthermore, blocking the conversion of sorbitol to fructoseby SDH mutation, which led to higher level of sorbitol accumulationand reduced oxidative stress, exacerbated cataract development(17). Taken together, these results strongly indicate that osmoticstress is the major contributing factor in diabetic cataractdevelopment in this experimental model in which cataract developsin a matter of weeks. This model simulates the acute diabeticcataract in patients with uncontrolled hyperglycemia. In patientswith diabetes and moderately well-controlled blood glucose level,cataract may take >10 yr to develop. It is likely that inthe slow-developing diabetic cataract, chronic oxidative stressmay be a more important factor. In the nerve, although the levelof sorbitol is increased during hyperglycemia, it is most likelynot the cause of diabetes-induced functional impairment. Thesorbitol level in the nerve of nondiabetic SDH-deficient miceis higher than that of diabetic wild-type mice (14), yet theNCV of the nondiabetic SDH-deficient mice is normal, indicatingthat a higher level of sorbitol alone does not cause any damageto the nerve. Polyol pathway–induced oxidative stressis most likely an important contributing factor to diabeticneuropathy. This is supported by a number a studies that showedthat antioxidant treatment significantly attenuated some ofthe symptoms of this disease (22–24).
Acknowledgments
This work was supported by Hong Kong RGC Grants HKU360/94M,HKU7259/98M, and HKU7259/00M to Dr. S.S.M. Chung and HKU7225/97Mto Dr. S.K. Chung
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Top
Abstract
Introduction
B (3), and JNK stress-associated kinases (4), and so forth. Inappropriate activation of these important regulatory molecules would have deleterious effects on cellular functions, and it is thought to contribute to the pathogenesis of various diabetic complications (5). However, it is not clear how hyperglycemia leads to increased oxidative stress. It is most likely the combined effects of increased levels of reactive oxygen species (ROS) and decreased capacity of the cellular antioxidant defense system. Glucose auto-oxidation (6), nonenzymatic glycation (7), and the interaction between glycated products and their receptors (8), overproduction of ROS by mitochondria (9), and the polyol pathway (10,11) all are potential sources of hyperglycemia-induced oxidative stress. This report focuses on the contribution of the polyol pathway to oxidative stress. 
-Tocopherol treatment prevents glomerular dysfunctions in diabetic rats through inhibition of protein kinase C-diacylglycerol pathway. Biofactors 7: 69–76, 1998[Medline]
