Diabetes causes increased oxidative stress in various tissues as 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), transcription factor NF-κB (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.
The polyol pathway consists of two enzymes. The first enzyme, aldose reductase (AR), reduces glucose to sorbitol with the aid of its co-factor NADPH, and the second enzyme, sorbitol dehydrogenase (SDH), with its co-factor NAD+, converts sorbitol to fructose. In animal models, treatment with AR inhibitors (ARI) was shown to be effective in preventing the development of various diabetic complications, including cataract, neuropathy, and nephropathy (12). It was thought that osmotic stress, from the accumulation of sorbitol, leads to diabetic lesions (13). Although this model may be applicable to the lens, in other tissues, such as sciatic nerve, the level of sorbitol does not correspond to the severity of neural dysfunction (14), suggesting that other mechanisms may be more important in contributing to diabetic lesions. Treatment of diabetic rats with an ARI attenuated the reduction of GSH in their lenses, suggesting that AR activity causes oxidative stress (15). However, the ARI may have free radical scavenging function; therefore, the normalization of GSH may not be due to the inhibition of AR (16). Here, we report the use of a genetic approach to demonstrate that both AR and SDH contribute to diabetes-induced oxidative stress.
Polyol Pathway and Diabetes-Induced Oxidative Stress in the Lens
Mice have low levels of AR in their lenses, and they are resistant to develop diabetic cataract. To determine the role of AR in the pathogenesis of cataract, we developed transgenic mice that overexpress the human AR cDNA specifically in their lenses. Expression of the AR transgene was found only in the lens and no other tissues. Under normal rearing condition, no morphologic abnormality was detected in the lenses of the transgenic mice, indicating that overexpression of AR per se does not have any deleterious effect on the lens. When induced to become diabetic by streptozotocin injection, the transgenic mice developed cataract at a rate proportional to the level of AR expression in their lenses, indicating that AR is the key enzyme in the pathogenesis of diabetic cataract (17).
These lens-specific AR transgenic mice were used to determine whether the polyol pathway activity contributes to diabetes-induced oxidative stress (18). When the wild-type mice were induced to become diabetic, their lenses showed no sign of experiencing oxidative stress. However, the lenses of diabetic transgenic mice had significant decrease in GSH level and significant increase in the level of malondialdehyde (MDA), indicative of oxidative stress (Figure 1). These results indicate that AR is the major contributor to diabetes-induced oxidative stress in the lens. Introducing a copy of the SDH-deficient mutation into the AR transgenic mice partially normalized the GSH and MDA levels in the diabetic transgenic mice, indicating that SDH also contributes to 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 neuropathy as indicated by reduced nerve conduction velocity (NCV) and signs of structural abnormality of the nervous tissues (19). To determine the role of polyol pathway in the pathogenesis of this disease, we developed AR gene knockout mice (20). The growth rate and reproductive capacity of these mice were similar to that of the wild-type mice. The only observable abnormality in the AR-deficient mice is that they drink and urinate more than the wild-type mice, indicating a mild impairment in their urine concentrating ability. However, this does not affect the levels of various electrolytes in their serum. When these mice were induced to become diabetic, they showed no reduction in their NCV, indicating that AR deficiency confers to these mice resistance to develop diabetic neuropathy (Figure 2). Whereas the diabetic wild-type mice showed significant reduction in the GSH level in their sciatic nerve, diabetic AR null mice showed no change in the GSH level, indicating that the polyol pathway is the major source of diabetes-induced oxidative stress in 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.
Discussion
We have shown that the polyol pathway is the major source of diabetes-induced oxidative stress in lens and the nerve. There are three potential mechanisms for the polyol pathway to contribute to oxidative stress (Figure 3). (1) AR activity depletes its co-factor NADPH, which is also required for glutathione reductase to regenerate GSH. Under hyperglycemic condition, as much as 30% of the glucose is channeled into the polyol pathway (10), causing a substantial depletion of NADPH and consequently a significant 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 stress because 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. Because fructose and its metabolites fructose-3-phosphate and 3-deoxyglucosone are more potent nonenzymatic glycation agents than glucose, the flux of glucose through the polyol pathway would increase advance glycation end products (AGE) formation. AGE, as well as binding of AGE to their receptors, are known to cause oxidative stress.
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 both the lens and the nerve, its role in the development of diabetic lesion in these two tissues seemed to be different. Osmotic stress, from the accumulation of sorbitol, is a more important factor for the development of diabetic cataract. This was demonstrated by the fact that administration of vitamin E and vitamin C, even though significantly normalized GSH and MDA levels in the diabetic 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 fructose by SDH mutation, which led to higher level of sorbitol accumulation and reduced oxidative stress, exacerbated cataract development (17). Taken together, these results strongly indicate that osmotic stress is the major contributing factor in diabetic cataract development in this experimental model in which cataract develops in a matter of weeks. This model simulates the acute diabetic cataract in patients with uncontrolled hyperglycemia. In patients with diabetes and moderately well-controlled blood glucose level, cataract may take >10 yr to develop. It is likely that in the slow-developing diabetic cataract, chronic oxidative stress may be a more important factor. In the nerve, although the level of sorbitol is increased during hyperglycemia, it is most likely not the cause of diabetes-induced functional impairment. The sorbitol level in the nerve of nondiabetic SDH-deficient mice is higher than that of diabetic wild-type mice (14), yet the NCV of the nondiabetic SDH-deficient mice is normal, indicating that a higher level of sorbitol alone does not cause any damage to the nerve. Polyol pathway–induced oxidative stress is most likely an important contributing factor to diabetic neuropathy. This is supported by a number a studies that showed that antioxidant treatment significantly attenuated some of the symptoms of this disease (22–24).
This work was supported by Hong Kong RGC Grants HKU360/94M, HKU7259/98M, and HKU7259/00M to Dr. S.S.M. Chung and HKU7225/97M to Dr. S.K. Chung
1. Konishi H, Tanaka M, Takemura Y, Matsuzaki H, Ono Y, Kikkawa U, Nishizuka Y: Activation of protein kinase C by tyrosine phosphorylation in response to H2O2. Proc Natl Acad Sci U S A 94: 11233–11237, 1997
2. Koya D, Haneda M, Kikkawa R, King GL: d-α-Tocopherol treatment prevents glomerular dysfunctions in diabetic rats through inhibition of protein kinase C-diacylglycerol pathway. Biofactors 7: 69–76, 1998
3. Mohamed AK, Bierhaus A, Schiekofer S, Tritschler H, Ziegler R, Nawroth PP: The role of oxidative stress and NF-κB activation in late diabetic complications. Biofactors 10: 157–167, 1999
4. Ho FM, Liu SH, Liau CS, Huang PJ, Lin-Shiau SY: High glucose-induced apoptosis in human endothelial cells is mediated by sequential activations of c-Jun NH2-terminal kinase and caspase-3. Circulation 101: 2618–2624, 2000
5. Rosen P, Nawroth PP, King G, Moller W, Tritschler HJ, Packer L: The role of oxidative stress in the onset and progression of diabetes and its complications: A summary of a Congress Series sponsored by UNESCO-MCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev 17: 189–212, 2001
6. Wolff SP, Dean RT: Glucose autoxidation and protein modification. The potential role of ’autoxidative glycosylation’ in diabetes. Biochem J 245: 243–250, 1987
7. Mullarkey CJ, Edelstein D, Brownlee M: Free radical generation by early glycation products: A mechanism for accelerated atherogenesis in diabetes. Biochem Biophys Res Commun 173: 932–939, 1990
8. Schmidt AM, Hori O, Brett J, Yan SD, Wautier JL, Stern D: Cellular receptors for advanced glycation end products. Implications for induction of oxidant stress and cellular dysfunction in the pathogenesis of vascular lesions. Arterioscler Thromb 14: 1521–1528, 1994
9. Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M: Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404: 787–790, 2000
10. Cheng HM, Gonzalez RG: The effect of high glucose and oxidative stress on lens metabolism, aldose reductase, and senile cataractogenesis. Metabolism 35: 10–14, 1986
11. Greene DA, Stevens MJ, Obrosova I, Feldman EL: Glucose-induced oxidative stress and programmed cell death in diabetic neuropathy. Eur J Pharmacol 375: 217–223, 1999
12. Oates PJ, Mylari BL: Aldose reductase inhibitors: Therapeutic implications for diabetic complications. Expert Opin Investig Drugs 8: 2095–2119, 1999
13. Kinoshita JH, Nishimura C: The involvement of aldose reductase in diabetic complications. Diabetes Metab Rev 4: 323–337, 1988
14. Ng TF, Lee FK, Song ZT, Calcutt NA, Lee AY, Chung SS, Chung SK, Ng DT, Lee LW: Effects of sorbitol dehydrogenase deficiency on nerve conduction in experimental diabetic mice. Diabetes 47: 961–966, 1998 [published erratum appears in Diabetes 47: 1374, 1998]
15. Gonzalez AM, Sochor M, McLean P: The effect of an aldose reductase inhibitor (Sorbinil) on the level of metabolites in lenses of diabetic rats. Diabetes 32: 482–485, 1983
16. Williamson JR, Chang K, Frangos M, Hasan KS, Ido Y, Kawamura T, Nyengaard JR, van den EM, Kilo C, Tilton RG: Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 42: 801–813, 1993
17. Lee AY, Chung SK, Chung SS: Demonstration that polyol accumulation is responsible for diabetic cataract by the use of transgenic mice expressing the aldose reductase gene in the lens. Proc Natl Acad Sci U S A 92: 2780–2784, 1995
18. Lee AY, Chung SS: Contributions of polyol pathway to oxidative stress in diabetic cataract. FASEB J 13: 23–30, 1999
19. Yagihashi S, Yamagishi SI, Wada RR, Baba M, Hohman TC, Yabe-Nishimura C, Kokai Y: Neuropathy in diabetic mice overexpressing human aldose reductase and effects of aldose reductase inhibitor. Brain 124: 2448–2458, 2001
20. Ho HT, Chung SK, Law JW, Ko BC, Tam SC, Brooks HL, Knepper MA, Chung SS: Aldose reductase-deficient mice develop nephrogenic diabetes insipidus. Mol Cell Biol 20: 5840–5846, 2000
21. Morre DM, Lenaz G, Morre DJ: Surface oxidase and oxidative stress propagation in aging. J Exp Biol 203: 1513–1521, 2000
22. Cameron NE, Tuck Z, McCabe L, Cotter MA: Effect of the hydroxyl radical scavenger, dimethylthiourea, on peripheral nerve tissue perfusion, conduction velocity and nociception in experimental diabetes. Diabetologia 44: 1161–1169, 2001
23. Kishi Y, Schmelzer JD, Yao JK, Zollman PJ, Nickander KK, Tritschler HJ, Low PA: Alpha-lipoic acid: Effect on glucose uptake, sorbitol pathway, and energy metabolism in experimental diabetic neuropathy. Diabetes 48: 2045–2051, 1999
24. Stevens MJ, Obrosova I, Cao X, Van Huysen C, Greene DA: Effects of DL-alpha-lipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy. Diabetes 49: 1006–1015, 2000
