Sugar Cataractogenesis Research Articles

Apoptotic cell death in the lens epithelium of rat sugar cataract

Apoptosis of lens epithelial cells (LECs) is implicated in the pathogenesis of several types of cataract formation. The high intracellular levels of polyol induce histological change in the LECs, which is considered the earliest event in sugar cataractogenesis. This study was designed to investigate whether high galactose exposure induces apoptosis in LECs during the development of sugar cataract. The effect of an aldose reductase inhibitor, SNK-860, was also examined. We induced sugar cataract in Sprague-Dawley rats by feeding them a 50% galactose-containing diet with or without SNK-860. The percentage of LECs undergoing apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) method, and DNA fragmentation analyses were performed. Galactitol levels in the lens epithelium were quantified by gas chromatography. The number of TUNEL-positive cells gradually increased throughout the period of galactose exposure, up to 5 days. DNA fragmentation analysis in LECs of rats fed a galactose-rich diet demonstrated an apparent ladder pattern. SNK-860 reduced the percentage of TUNEL-positive cells, the amount of intracellular galactitol, and the levels of DNA laddering. To explore the mechanism of the apoptotic process, the expression of p53, a potent mediator of apoptosis, was examined. Based on Western blot and real-time reverse transcription-polymerase chain reaction results, the amount of p53-expression increased at both the protein and mRNA levels after galactose exposure, and the increase in p53-expression was inhibited by SNK-860. Based on these results, we concluded that apoptosis occurs in rat lens epithelial cells following galactose exposure. Furthermore, the reduction of apoptosis by aldose reductase inhibitor suggests that this apoptosis is associated with the accumulation of sugar alcohols. It is probable that the mechanism of apoptosis during sugar cataract formation involves the increased expression of p53.

Effects of Cataractogenesis on the CDP-Choline Pathway: Changes in ATP Concentration and Phosphocholine Synthesis during and after Exposure of Rat Lenses to Sugars in vitro and in vivo

We measured choline influx and phosphorylation, ATP concentration ([ATP]), choline kinase activity and lens swelling during formation and partial reversal of sugar cataracts in rat lenses incubated with xylose or galactose and in lenses of galactosemic rats. [ATP] and phosphocholine (P-Cho) synthesis decreased about 60 and 40% after 4 h in normal rat lenses incubated up to 24 h in medium containing 30 mM xylose and partially recovered when the lenses were then removed from the xylose. Incubation with the somewhat less cataractogenic sugar galactose decreased P-Cho synthesis but had little effect on [ATP]. P-Cho synthesis decreased rapidly in the lenses of rats fed a 50% galactose diet, but began recovery by the third day on this diet. [ATP] decreased for at least 10 days during the galactose diet and did not recover, even with resumption of the control diet (50% starch) after 4 or 7 days. The results of in vitro and in vivo sugar cataractogenesis differed from each other in several respects, including effects on choline influx and the degree to which the changes were reversible. The in vitro and in vivo sugar cataracts, however, could both produce swelling and opacification of the lens and decreased P-Cho synthesis and [ATP]. Neither model caused a substantial change in the choline kinase activity (as measured in cell-free assays). The data did not generally support the hypothesis that decreased [ATP] causes decreased P-Cho synthesis.

Regulation of aldose reductase by aldehydes and nitric oxide.

Aldose reductase (AR) is a member of the aldo-keto reductase superfamily. It was initially discovered in the seminal vesicles, where it is believed to catalyze the NADPH-mediated reduction of glucose to sorbitol-which in turn is metabolized to fructose required for spermatogenesis (for review see Bhatnagar and Srivastava, 1992). The conversion of glucose to fructose via sorbitol, or the so called polyol pathway was later identified in several other tissues and AR was found to be universally expressed in most tissues examined and in species ranging from yeast to man. Based on the observations that high concentration of polyols accumulate during sugar cataractogenesis, it was suggested that during hyperglyce- mia, the flux of glucose via the polyol pathway is increased and that due to the low permeability of the membrane to polyols, sugar alcohols accumulate in diabetic tissues leading to marked hydration, cell swelling and lysis (Kinoshita and Nishimura, 1988).

Effects of Xylose on Monkey Lenses in Organ Culture: A Model for Study of Sugar Cataracts in a Primate

Lenses exposed to high concentrations of xylose in organ culture produce xylitol, and they lose transparency and exhibit other changes characteristic of cataracts. Most previous studies of this model system for cataractogenesis have employed rat or rabbit lenses, where the activity of the enzyme aldose reductase has been definitely implicated as the initiating factor. Since lenses from this species have much higher aldose reductase activity and have other differences relative to human lenses, the relevance of these findings to the human lens is uncertain. To determine the effects of xylose on the lenses of a primate, lenses from the rhesus monkey(Macaca mulatta)were incubated 24–48 hr in control medium or in TC-199 medium containing 30 mmxylose. Xylose caused a general haziness, focal swelling of epithelial cells, and swollen peripheral fiber cells, but the changes were much less pronounced than in rat lenses under similar conditions. Monkey lenses exposed to 30 mmglucose, galactose or xylose accumulated measurable sorbitol, dulcitol or xylitol, respectively, but the amounts were much lower than in rat lenses, perhaps reflecting the lower aldose reductase and higher sorbitol dehydrogenase activities in monkey lenses. The damage to monkey lenses appeared to be limited to the outer layers. In monkey lenses, xylose caused little, if any, change in membrane transport of choline or α-aminoisobutyrate, but severely depressed synthesis of phosphorylcholine (P-choline), and increased leakage of P-choline into the culture medium, leading to a decrease in the P-choline concentration within 24–48 hr. In summary, xylose-induced damage to monkey lenses in organ culture is qualitatively similar to that seen in rat lenses, but the changes are much less rapid and severe. Culture of monkey lenses with xylose provides a model system to extend previous studies of sugar cataractogenesis in rats to a species more closely related to humans.

Taurine prevents galactose-induced cataracts

Intact lenses from New Zealand white rabbits were incubated in tissue culture media containing either 5 mM glucose or 5 mM glucose plus 30 mM galactose. The standard media did not contain taurine. Lenses were also cultured in a third medium containing 30 mM galactose plus 0.2 mM taurine. The frequency of cataract formation was evaluated as a function of the culture media. One lens ( 1 10 ), in media containing 5 mM glucose, developed a lenticular opacification during a 72-h incubation. Lenses ( 12 15 ) incubated in 30 mM galactose, without taurine, developed cataracts; fewer lenses ( 2 13 ) exposed to 30 mM galactose plus 0.2 mM taurine developed cataracts ( p < 0.005). Galactose cataracts have been associated with lens edema attributed to the osmotic stress of tissue polyol (galactitol) accumulation. The water content of the noncataractous and cataractous lenses in this experiment did not differ. Lens edema, therefore, was not thought to be important in cataract pathogenesis. Taurine, an organic osmolyte was lower (5.1 ± 1.5 μmol/g protein) in cataractous lenses than in control lenses (10.0 ± 1.0 μmol/g protein). Malondialdehyde, an indicator of lipid peroxidation, was higher (36.6 ± 5.0 μmol/g protein) in lens-containing opacifications than in noncataractous lenses (10.1 ± 1.9 μmol/gm protein) ( p < 0.01). The levels of malondialdehyde suggest that lipid peroxidation was increased in the process of sugar cataractogenesis. The malondialdehyde content of all the lenses correlated inversely ( r = −0.53, p < 0.01) with the coincident lens taurine levels. Taurine appears to protect the lens against the development of sugar cataracts; its inverse relationship with lens malondialdehyde suggests this is an antioxidant effect.

The role of lens epithelium in sugar cataract formation

Previous evidence has shown clearly that sugar cataract formation results from unusually high intracellular levels of polyol. Documentation of polyol-related histological changes in the cortical fiber cells of the equatorial zone has been extensive. However, little attention has been given to the early changes in the lens epithelial cells, in spite of the fact that the highest level of aldose reductase is found in this layer of the lens. Also, cultured lens epithelial cells exposed to high sugar levels exhibit rapid accumulation of polyol and show ultrastructural alterations. Therefore, a study was designed to evaluate the role of the lens epithelium in sugar cataract formation. Specifically, an attempt was made to localize the earliest fine structural lesions in intact lenses of galactose-fed rats and to test their relation to aldose reductase. Rats were fed either a normal diet or a 50% galactose diet with or without sorbinil, an aldose reductase inhibitor. Rats were killed at varying periods of time ranging from 6 to 96 hr, and the eyes were processed for light and electron microscopy. The first detectable abnormalities occurred after 36 hr of galactose feeding, and were limited to the central lens epithelium. Cell edema, apparent dilution of cytoplasm, rounding of nuclei, aberrant intracellular vacuoles, and loss of normal tortuosity of cell boundaries were the salient lesions. No changes were detectable in the equatorial zone until 48 hr, and no deviation from the control structure was found in any of the rats treated with an aldose reductase inhibitor. These studies clearly show that the epithelium is the area of the lens first affected. Since the lens epithelium is the principal site of important regulatory mechanisms, the finding probably explains why the active uptake mechanisms are affected early in sugar cataractogenesis, and suggests that the epithelium may be more involved in sugar cataractogenesis than previously suspected.

Nonosmotic Diabetic Cataracts

It has been suggested that sugar cataracts associated with diabetes mellitus result from the accumulation of excess sorbitol within lens fibrils. Swelling of lens fibrils occurs when water moves in to maintain osmotic balance; the excess water causes disruption of fibrils and cataract formation. Other studies have indicated that more than sorbitol-induced osmotic stress is involved. Our study used lenses collected from rats after 21 or 44 d of streptozotocin diabetes. Cataracts formed in untreated 44-d streptozotocin diabetic rats, but were not apparent in the 21-d untreated diabetic animals. Lens sorbitol increased in the diabetic animals both before and after cataract formation. Lens taurine varied inversely with the sorbitol content in a fashion that resulted in no net change in total lens osmoles. Lens water did not increase in the diabetic animals with or without cataracts. The aldose reductase inhibitor Sorbinil prevented the increase in lens sorbitol in both the 21- and 44-d streptozotocin diabetic rats; cataract formation was prevented in the 44-d diabetic animals. The lens water in untreated diabetic animals with cataracts did not differ from lens water in the Sorbinil-treated diabetic animals that did not develop cataracts. Sorbinil treatment of diabetic animals was associated with normalization of both lens sorbitol and taurine levels. Taurine has been shown to serve both as an osmoregulator and as an antioxidant. The apparent increase in lens osmolality attributed to sorbitol was counterbalanced by an equimolar reduction in taurine concentration. The reciprocal relationship between taurine and sorbitol reduces the likelihood of an osmotic mechanism for sugar cataractogenesis; the reduced lens taurine, however, may increase the risk of lens protein oxidation and subsequent cataract formation. Thus in vivo sugar cataract formation may be an oxidative process rather than an osmotic phenomenon.

Regional distribution of calcium in alloxan diabetic rabbit lens.

A diabetic rabbit model was developed for investigation of cataractogenesis and other changes in the anterior segment. Rabbits were fasted, injected with 0.7 mg/kg alloxan, fed 1% glucose solution for 24 hrs and returned to a normal diet. Animals showing and maintaining blood glucose of greater than 300 mg% within two days were used in this study. Concomitant with increase in blood glucose was a rise in aqueous humor glucose and osmolality, together with a decrease in ascorbate concentration. Vacuoles and small discrete opacities developed, and in some cases, at longer time periods complete opacity of anterior or posterior aspects was found. Total calcium content of the whole lens increased up to 2-fold, especially after 60 days, and was correlated with a decrease in lens transmittance of a He/Ne laser beam and also with high osmolality of the aqueous humor. Free calcium was six-fold higher in opaque areas than clear areas, and was 100-fold higher in vacuoles. It is suggested that, in addition to the recognized role in sugar cataractogenesis of osmotic stress due to sorbitol accumulation in the lens, changes of intracellular calcium in localized areas of the lens and stresses imposed by changes in aqueous humor osmolality may also be important.

Modelling cortical cataractogenesis: VI. Induction by glucose in vitro or in diabetic rats: Prevention and reversal by glutathione

Cataractogenesis can be induced by glucose in the rat lens in vitro and in vivo. In vitro, this is done by increasing the amount of glucose in the medium surrounding the isolated lens; within 48 hr considerable globular degeneration is seen subcapsularly, deeper in the equatorial region. In vivo, it is achieved by making the rat diabetic by injecting streptozotocin i.v., which selectively destroys the β-cells of the pancreas; the blood serum glucose level increases markedly, and thence the aqueous humour level and, in turn, the lens concentration. Globular degeneration occurs as in vitro, but not until 6 weeks is a degree of damage observed comparable to that seen in the lenses incubated in vitro for only 48 hr. Lenticular sorbitol and fructose are also markedly elevated as a result of the high glucose levels. If glutathione (GSH) is present in the medium (0·1 m m) or injected s.c. daily into the diabetic rats, there is no evidence of subcapsular globular degeneration of the cortical fiber cells, even though the lenticular levels of glucose, sorbitol and fructose are the same as when GSH was not given; this is true for either the in vitro or in vivo situation. although individual values in the two situations do differ somewhat from one another. When rats were given GSH beginning several weeks after the diabetic state had been induced, the damage subsequently observed was much less than if the rats had been diabetic without GSH for the same total length of time; it was also much less than damage which should have occurred by the time GSH treatment was instituted. It would thus appear that a certain amount of reversal of the globular degeneration is possible, although damage in the equatorial region (wedge-shaped) seems less amenable to rescue by glutathione. The data indicate that glutathione can prevent or diminish the severity of sugar cataractogenesis. and that there would appear to be more steps in sugar cataractogenesis than simply osmotic damage, although this may be the primary event.

Diminished sugar cataractogenesis by quercetin

Aldose reductase has been implicated in the pathogenesis of sugar cataracts, therefore inhibition of this enzyme may represent a pharmacological approach for the delay and possible prevention of cataractogenesis induced by diabetes or glactosemia. In the neonatal rat, the fused eyelids provide a natural delivery chamber, an orbital pouch, for the daily deposition of an aldose reductase inhibitor. Topical administration of quercetin; a pentahydroxyflavone, to the orbital pouch of the galactosemic neonatal rat diminishes cataractogenesis in the corresponding lens. Comparison with the contralateral lens, indicates that quercetin (1 × 10 −5 m) reduces intracellular edema, prevents extracellular fluid accumulation and maintains cellular interdigitation of the superficial anterior cortical fiber. In addition to preserving fiber integrity, topical application of quercetin maintains lens growth as evidenced by radius and dry weight measurements. In normal neonates, quercetin (1 × 10 −5 m) does not alter fiber structure or development. Dietary administration of quercetin to the galactosemic neonates is also effective in maintaining individual fiber thickness, density and interdigitations. Therapeutically, many ophthalmic compounds are administered in the form of drops or soluble ophthalmic inserts, therefore the preclinical protocol for this study encompasses the basic correlates of clinical use; the neonatal orbital pouch may become a useful model to further investigate the activity and potency of new ophthalmic agents.

The absence of cataracts in mice with congenital hyperglycemia

Investigations were conducted to determine the biochemical basis of the absence of cataracts in genetically diabetic mice. The resistance of these mice to cataracts appears to be due to the low activity of aldose reductase in the lens. Consequently only low amounts of polyols accumulate even after persistent hyperglycemia. The finding thus lends further support to the polyol theory of sugar cataractogenesis. The accumulation of sugar alcohols in galactosemic mice lenses was also much lower as compared to that in the lenses of galactosemic rats. A study was made of the enzymes that influence the polyol level in the lenses of various mouse strains. The activities of aldose reductase, polyol dehydrogenase, hexokinase and glucose-6-phosphate and 6-phosphogluconate dehydrogenases in the mouse and rat lenses were determined. The activities of all the enzymes except that of aldose reductase were similar in the lenses of both species. The aldose reductase activity in the mouse lens was only one tenth of that present in the rat lens.