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Research Communications |
a Institute of Molecular Biology, University of Hong Kong, Pokfulam, Hong Kong, People's Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSREFERENCES |
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Key Words: aldose reductase • sorbitol dehydrogenase • glutathione • malondialdehyde • transgenic mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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There is accumulating evidence, however, showing the contribution of oxidative stress to the development of diabetic cataract (12–15). A loss of antioxidants, like vitamin C (VC), vitamin E (VE), and glutathione, were found in lenses under hyperglycemic condition (16, 17). Supplementation of antioxidants such as butylated hydroxytoluene (18, 19), Trolox (20), and VE (21) have been shown to be able to retard or ameliorate the development of diabetic and galactosemic cataracts. However, some reports also showed the absence of any beneficial effects (22, 23). To date, the actual source of oxidative stress during diabetes is still not clear, but the elevation of blood glucose is probably involved (24, 25). It has been suggested that glucose autoxidation and nonenzymatic glycation, together termed glycoxidation, are the major contributors to the increase in free radicals in diabetic lens (26, 27). During diabetes, the antioxidation system in the lens may also be compromised. Reduced glutathione (GSH) was found to be depleted in diabetic lenses (28, 29), which was accompanied by an increase in the level of lipid peroxidation products (LPO). With the administration of ARIs, both GSH and LPO were restored to near normal levels (28, 30), indicating the involvement of AR in the generation of oxidative stress. However, the specificity of these chemical inhibitors cannot be ascertained, and many ARIs are also potential free radical scavengers. Recently, the conversion of sorbitol to fructose via sorbitol dehydrogenase (SD) has also been suggested to contribute to redox imbalance in diabetic tissues (31, 32). SD inhibitors (SDIs) were shown to attenuate the diabetes-induced increase in cytosolic NADH/NAD+ ratio in diabetic retina (32), implying that SD may also contribute to diabetic cataract. In this study, we made use of mouse mutants to study the relationship between polyol pathway and oxidative stress, and the contribution of oxidative stress to diabetic cataract.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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A-crystallin promoter and SV40 polyadenylation sequence. The hybrid DNA was injected into the pronucleus of fertilized mouse eggs. Mice from the transgenic line CAR648, heterozygous for the transgene, were used in this study. The lens AR level of CAR648 and normal mouse are 133.7 ± 8.5 and 1.4 ±0.094 nmol NADPH/(min·mg protein), respectively.
Diabetes induction
Mice (3- to 4-wk old, 15–20 g) were made diabetic by streptozotocin (STZ) injection at a single dose of 200 mg/kg body weight. STZ solution was prepared immediately before injection by dissolving the powder in 0.1 M citrate buffer, pH 2.5. Animals of the control group were injected with the same volume of the vehicle. Blood glucose level was monitored with glucose test strips (HaemoGluko test, Boehringer Mannheim, U.K.), and animals with blood glucose level lower than 500 mg/dl were excluded from the experiment.
Buthionine sulfoximine treatment
BSO (L-buthionine-[S,R]-sulfoximine) was obtained commercially (Sigma, St. Louis, Mo.). Stock solution of BSO was prepared by dissolving the powder in 0.1 M NaCl solution immediately before use. Mice were injected intraperitoneally with BSO solution twice daily (4 µmol/g body weight) for 7 days to maintain a low GSH level.
Lens culture in glyceraldehyde-containing medium
Lenses from transgenic mouse line CAR648 were isolated and cultured in vitro as described previously (33). Briefly, contralateral lenses were cultured individually in M199 basic medium (Gibco, Gaithersburg, Md.) for 24 h. Lenses that remained clear were then switched to either one of the following media: medium A, basic M199, medium B, basic M199 supplemented with 3 mM DL-glyceraldehyde, or medium C, basic M199 supplemented with 3 mM DL-glyceraldehyde + 0.2 mM ARI AL1576. The lenses were cultured in a 5% CO2 incubator at 37°C for 5 days, and lens morphology was observed daily. At the end of the experiment, lenses were harvested and subject to GSH and malondialdehyde (MDA) measurements.
Treating mice with vitamin C
Vitamin C, in the form of ascorbic acid (Sigma) mixed into pulverized rat chow, was fed to mice. CAR648 transgenic mice were divided into three groups and treated as follows: group 1: untreated nondiabetic; group 2: STZ-induced diabetes; group 3: STZ-induced diabetes, with 0.1% (w/w) VC supplement in pulverized rat chow.
Treating mice with vitamin E
Vitamin E, in the form of tocopherol solution (Sigma), was dissolved in soybean oil immediately before use. Transgenic mice from line CAR648 were divided into three groups and treated as follows: group 1: untreated nondiabetic; group 2: STZ-induced diabetes; group 3: STZ-induced diabetes, with daily subcutaneous injection of VE at a dose of 960 i.u./kg body weight.
Cataract monitoring
Mouse pupil was dilated with 1% tropicamide (Alcon, Puurs, Belgium) and the lens was monitored daily with a slit-lamp microscope. The formation of cataract was recorded by photography.
Determination of lens sorbitol content
Lens sorbitol level was determined as previously described (11). Briefly, mouse lenses were isolated by posterior approach, homogenized, and deproteinized. The supernatant was lyophilized and resuspended in pyridine. The polyols were derivatized by phenylisocyanate and separated by reverse-phase high-performance liquid chromatography. The elution was then monitored spectrophotometrically at 240 nm.
Determination of glutathione
Lens glutathione in reduced form (GSH) was assayed by reacting with dithionitrobenzoic acid (DTNB) as described (34). Briefly, mouse lenses were homogenized in cold 20 mM EDTA solution on ice. After deproteinization with 5% trichloroacetic acid, an aliquot of the supernatant was allowed to react with 150 µM DTNB. The product was detected and quantified spectrophotometrically at 416 nm. Pure GSH was used as standard for establishing the calibration curve.
Determination of lipid peroxidation products
The level of LPO in the lens was assayed by measuring the reaction products between MDA and thiobarbituric acid (TBA), as described (35), with modifications. Briefly, mouse lenses were homogenized in cold phosphate buffer on ice. LPO in the crude extract was precipitated and washed with phosphotungstic acid. After resuspending in distilled water and adjusting the pH to 3, LPO was allowed to react with TBA at 95°C for 1 h. The reaction mixture was then cooled down to room temperature and the reaction product was extracted with equal volume of n-butanol. The product was measured fluorometrically with excitation wavelength at 515 nm and emission wavelength at 553 nm. Tetramethoxypropane (TMP) was used as standard for setting up the calibration curve.
RESULTS
GSH and MDA content in lenses of diabetic transgenic mice overexpressing aldose reductase
To study whether AR is involved in the generation of oxidative stress associated with diabetes, the levels of GSH and MDA, a product of lipid peroxidation, in the lenses of diabetic AR-overexpressing transgenic mice and their nontransgenic littermates were measured. Mice heterozygous for the transgene from the line CAR648 (11) and their nontransgenic littermates were divided into two groups. In the experimental group, diabetes was induced with streptozotocin, whereas the control group was treated with the vehicle only. Under normal conditions, there were high levels of GSH in the lenses of both CAR648 transgenic mice and their nontransgenic counterparts (
Fig. 1a).
In the hyperglycemic state, however, a precipitous drop in the GSH level in transgenic mouse lenses was observed. The decrease in GSH level was accompanied by a twofold increase in lens MDA level ( Fig. 1b), suggesting that the depletion of GSH leads to an increase in oxidative stress. There was no significant change in GSH and MDA content in the lenses of diabetic nontransgenic counterparts, suggesting that hyperglycemic oxidative stress in the lens is mainly the result of polyol pathway activities.
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GSH content in transgenic mouse lenses cultured in glyceraldehyde-containing medium
Our previous results showed that the AR-overexpressing transgenic mice accumulated sorbitol to a high level in their lenses under hyperglycemic condition (11). The accompanying osmotic stress may cause leakage or impairment of import of amino acids, leading to reduced glutathione synthesis in the lens (16, 17, 36, 37). On the other hand, the accelerated glucose flux through the polyol pathway during diabetes may compete with glutathione reductase for the cofactor NADPH (12, 28), thus hindering the regeneration of GSH. To gain a better understanding on the mechanism of GSH depletion in diabetes, transgenic mouse lenses were incubated in culture medium containing glyceraldehyde, which has been shown to be a better substrate for AR than glucose (38). Unlike sorbitol, the resulting product glycerol tends not to accumulate in cells, but is further metabolized (39, 40); thus, the flux of glyceraldehyde through the polyol pathway would not generate a significant osmotic burden to the cells in lens. When lenses were cultured in medium containing glyceraldehyde for 5 days, the level of GSH in transgenic mouse lenses decreased to about the same levels as that of the lens in diabetic AR transgenic mice ( Fig. 2),
indicating that osmotic stress is not the major cause of GSH depletion. The decrease in lens GSH was prevented by the addition of a AR inhibitor AL1576 ( Fig. 2), which has previously been shown to be potent in inhibiting AR activity and preventing diabetic cataract formation in the transgenic mice (11).
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Involvement of sorbitol dehydrogenase in oxidative stress associated with diabetes
There is accumulating evidence showing that the conversion of sorbitol to fructose by SD may also contribute to redox imbalance, probably due to the depletion of its cofactor NAD+ (31, 32). To determine the involvement of SD in diabetes-induced oxidative stress, homozygous AR-overexpressing transgenic mice (AR+/+SD+/+) were crossed with mice deficient in SD (AR-/-SD-/-), previously characterized in our laboratory (41), generating mice heterozygous for the AR transgene, with 50% of the wild-type SD activity (AR+/-SD+/-). These mice were then induced to become diabetic by streptozotocin. Under normal physiological conditions, there is no significant difference in the lens GSH (
Fig. 1a) and MDA levels ( Fig. 1b) between the SD-deficient mice (AR+/-SD+/-) and those with normal level of SD (AR+/-SD+/+). Under hyperglycemic conditions, a decrease in lens GSH content ( Fig. 1a), accompanied by an elevation of lens MDA level ( Fig. 1b) was observed in SD-deficient mice as in the AR transgenic mice. However, in the lenses of SD-deficient mice, GSH level was reduced only by about 52% compared to that of the nondiabetic control, in contrast to the more than 70% reduction in AR-overexpressing transgenic mice. MDA in these SD-deficient mice was also accumulated to a lesser extent, to about 59% higher than the nondiabetic control, compared to the 120% increase in AR transgenic mice. Thus, a 50% reduction in SD activity significantly attenuated GSH loss and MDA buildup in the diabetic mouse lenses, indicating that SD also contributed significantly to hyperglycemic oxidative stress. Since SD deficiency leads to greater sorbitol accumulation and increased osmotic stress (11), these results also support the notion that GSH depletion is not due to leakage induced by osmotic stress.
Effects of vitamins C and E on diabetic cataract
Findings from the present study indicated that the both AR and SD in the polyol pathway are involved in the generation of oxidative stress associated with diabetes. The question is how significant is this polyol pathway-mediated redox imbalance contributes to the etiology of diabetic cataract. To address this question, diabetic AR-transgenic mice were subjected to VC or VE treatment, since both of these vitamins are known to have antioxidant effects. In the AR-overexpressing transgenic mice, the first sign of vacuole development at the periphery of the lens was observed 7 days after the initiation of hyperglycemia. With VC or VE treatment, the rate of progression of cataract was delayed. At 10 days after induction of diabetes, the number of vacuoles in the VC-treated ( Fig. 3c)
and VE-treated ( Fig. 3f) lenses was significantly less than that in the untreated diabetic group ( Fig. 3b, e). However, treatment with these antioxidants did not prevent cataract formation; it delayed the development of lens opacity only by 2–3 days.
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To determine whether the beneficial effect of vitamin C and E was mediated through the inhibition of AR activity instead of their antioxidation effects, sorbitol content in the diabetic mouse lenses was measured. In the lenses of VC-treated diabetic mouse, sorbitol was found to accumulate to a similar high level compared to the untreated diabetic counterpart ( Fig. 4).
The same finding was also observed in VE-treated diabetic mouse lenses 1 wk after the treatment ( Fig. 4). There was even a further elevation in the sorbitol level in the VE-treated diabetic mouse lenses at the end of the second week after induction of diabetes ( Fig. 4). Neither VC nor VE had any effect in restoring the GSH content in the lenses of diabetic transgenic mice ( Fig. 5a).
However, both vitamins served to reduce the formation of MDA in the diabetic mouse lens. The lenses of mice treated with VC and VE showed a 21% and 44% decrease in lens MDA level, respectively ( Fig. 5b). These results indicated that the beneficial effects of these vitamins were mediated through their antioxidant actions, not the inhibition of the polyol pathway.
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Depletion of GSH in mouse lenses with BSO
Since antioxidants can delay the development of diabetic cataract, we wanted to find out whether oxidative stress caused by the depletion of GSH would be sufficient to cause cataract. To deplete lens GSH content without causing osmotic stress, we treated normal mice with BSO. BSO is a chemical inhibitor directed against the enzyme 
-glutamylcysteine synthetase, which is involved in the synthesis of GSH (42, 43). The experimental group received BSO injection twice daily for 1 wk. However, no sign of cataract was observed in the BSO-treated mice throughout the course of experiment, even though there was an almost complete depletion of GSH in their lenses ( Fig. 6a).
The depletion of GSH in BSO-treated mouse lenses was accompanied by an elevation of the MDA level, reaching a level about 30% higher than that in the diabetic mouse lenses ( Fig. 6b). These results indicated that both GSH depletion and MDA accumulation by themselves were not able to induce cataract formation.
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DISCUSSION
Evidence obtained from clinical and animal studies suggested an association between oxidative stress and diabetes (44–49). However, both the source and the mechanism of its formation are still uncertain. Furthermore, it is not clear if oxidative stress is responsible for the development of diabetic complications. In this report, we demonstrated that the flux of glucose through the polyol pathway is the major source of diabetes-associated oxidative stress in the ocular lens. However, oxidative stress is only a minor contributing factor for diabetic cataract formation in this animal model.
It is surprising that diabetes does not induce significant oxidative stress in the lens of nontransgenic mice. Part of the reason is that the level of AR in the lens of these mice is very low (11). This finding indicates that the oxidative stress observed in the streptozotocin-treated AR-transgenic mice was not due to the oxidative effects of the drug itself, as suggested by earlier reports (50, 51). The major source of oxidative species in diabetic state is thought to be from the increase in glucose autoxidation and nonenzymatic glycation (26, 52, 53). However, the fact that the lenses of nontransgenic mice showed no increase in MDA in diabetic state indicates that the glycoxidation reaction does not contribute to oxidative stress in this tissue. This is similar to the findings in rat, in which administration of AR inhibitor to diabetic rats almost completely normalized the lens MDA levels (54). In those experiments, it is not clear if the residual increase in MDA in the presence of AR inhibitor is the result of incomplete inhibition of AR or it is the result of oxidative stress induced by glucose through glycoxidation or other means.
The competition between AR and glutathione reductase for the cofactor NADPH in the hyperglycemic state is most likely the cause of GSH depletion. However, it has been suggested that diabetes-associated GSH depletion may be secondary to the osmotic stress caused by sorbitol accumulation. Osmotic swelling of diabetic lens may render the cells leaky (55), enhancing the loss of GSH accumulated in the lens (29). Disrupted cell membrane by osmotic stress may also interfere with amino acid transport into the lens (36), and hence the biosynthesis of GSH (29). There were also reports showing that these changes occurred before the lens is exposed to osmotic stress (16, 17). For a better understanding of the cause of GSH loss, lenses of the AR-overexpressing transgenic mice were cultured in medium containing glyceraldehyde; so that the AR reduction activity is not accompanied by osmotic stress. The GSH level of these lenses was reduced to 50% of the untreated counterparts, indicating that GSH depletion is most likely due to NADPH depletion. This is also supported by the fact that SD deficiency, which causes greater osmotic stress, in fact led to an attenuation of the GSH loss in the lens of diabetic mice.
The reduction of glucose by AR is clearly the major cause of diabetic cataract in this transgenic mouse model. What is not clear is the relative contribution of osmotic stress and oxidative stress, which are both generated through the activity of AR, to cataract development. The question was addressed by using a GSH synthesis inhibitor BSO to deplete lens GSH without generating osmotic stress. The resulting dramatic drop in lens GSH content, which was accompanied by an elevation in LPO level, was even more severe than that in the lenses of the diabetic transgenic mice. However, no sign of cataract was observed in the BSO-treated mice. These results further demonstrate that oxidative insults alone may not be sufficient to be a causative factor in diabetic cataract. These results also show the inverse correlation between GSH level and LPO contents, similar to that observed in diabetes-induced oxidative stress, further confirming the important role of GSH as a cellular antioxidant for removing oxidative species (56).
Recently, there are accumulating evidence pointing to the pathological role of SD-mediated reduction in NAD+/NADH ratio during diabetes (31, 32). Our results from the SD-deficient mice also demonstrated the involvement of SD activity in hyperglycemia-induced oxidative stress in diabetic cataract. Accompanying the elevated glucose flux through the polyol pathway in diabetes, the oxidation of sorbitol to fructose via SD would also increase, resulting in a decrease in cytosolic NAD+/NADH ratio. To a certain extent, this redox balance is maintained in equilibrium by coupling to the reduction of pyruvate to lactate. Under hyperglycemic state, however, the large increase in NADH level tends to inhibit glycolysis at the step of the formation of 1,3-bisphosphoglycerate from glyceraldehyde-3-phosphate (57), thus limiting the availability of pyruvate for the regeneration of NAD+. This forms a vicious circle, leading to a further channeling of glucose into the polyol pathway due to the inhibition of glycolysis, which further perturbs the cytosolic NAD+/NADH ratio. By reducing the oxidation of sorbitol to fructose via the introduction of a SD-deficient mutation, AR-mediated GSH depletion and LPO accumulation were shown to be attenuated, probably through the relief of the inhibition of glycolysis so that less glucose was diverted into the polyol pathway. This hypothesis does not necessarily contradict our previous finding that an inhibition of SD activity led to a further accumulation of sorbitol in the lens (11), since sorbitol so formed was not further metabolized albeit the glucose flux through the pathway was reduced.
The involvement of oxidative insults in the pathogenesis of diabetic cataract was indicated by the delay in the progression of cataract development in mice treated with antioxidants VC and VE. The failure of these vitamins to normalize the lens sorbitol level in the diabetic mice illustrated that the beneficial effect was not mediated through the modulation of AR activity, as previously reported by Ross et al. (58). Neither VC nor VE rescued GSH depletion in the diabetic mouse lenses. Instead, a partial normalization of MDA content was found, with VE having a stronger effect than VC. These results indicated that VC and VE exerted their anticataractogenic effects through the removal of oxidative species to reduce the oxidative insults to the lens cells, resulting in a lowering of MDA in diabetic lenses. This helps to preserve membrane integrity, which may account for the additional increase in sorbitol content in diabetic lenses under VE treatment. The prevention of cell death, which leads to leakage of intracellular contents including sorbitol, may also be a mechanism for maintaining lens sorbitol concentration, as suggested by Ross et al. (58).
In conclusion, our findings showed that polyol pathway is the major contributor to the generation of hyperglycemic oxidative stress in the ocular lens, in addition to osmotic stress. AR-mediated oxidative insults by itself is not sufficient to be a causative factor in diabetic cataract. However, it should be pointed out that the AR transgenic mice used in this study develop diabetic cataract within 2–3 wk. This probably simulates those acute diabetic cataract in human. However, most diabetic cataract takes more than 10–20 years to develop. In the slow-developing cataract, the lenses are unlikely to experience a rapid and large increase in polyol to cause serious osmotic stress (59). Rather, the chronic oxidative insults from the polyol pathway activities could be a more important contributing factor. This hyperglycemia-induced oxidative stress is also likely to play an important role in the development of diabetic complications in tissues where sorbitol accumulation is far below than that observed in cataractous lens to pose any osmotic stress.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: AR, aldose reductase; ARIs, aldose reductase inhibitors; BSO, L-buthionine-[S,R]-sulfoximine; DTNB, dithionitrobenzoic acid; GSH, glutathione; LPO, lipid peroxidation products; MDA, malondialdehyde; SD, sorbitol dehydrogenase SDIs, SD inhibitors; STZ, streptozotocin; TBA, thiobarbituric acid; TMP, tetramethoxypropane; VC, vitamin C; VE, vitamin E.
Received for publication June 23, 1998.
Revision received August 31, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSREFERENCES |
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