Jin H. Kinoshita

Aldose reductase in diabetic complications of the eye

Aldose reductase (AR) appears to initiate the cataractous process in galactosemic and diabetic animals. Sugars in excess are converted to polyols by lens AR. In sugar cataracts, polyols accumulate to levels substantial enough to cause a hypertonicity leading to lens fiber swelling. All other changes appear secondary to polyol accumulation and lens swelling. The development of sugar cataracts can be duplicated in organ culture. In culture, the various changes that occur were minimized or did not occur when inhibitors of AR were included in the medium. Moreover, AR inhibitors were shown to effectively delay the onset of sugar cataract development in animals. A defect in the corneal epithelium of diabetics became apparent in vitrectomy. One manifestation of this problem was the delay in the reepithelialization of denuded corneas. In examining this problem experimentally, the epithelium was removed from the corneas of diabetic and normal rats. The regeneration of epithelium in corneas of diabetic rats required a longer period than in the normal. The possibility that AR, active in the epithelium, was involved in this phenomenon was investigated. The corneal epithelium was removed from both eyes of a diabetic rat. One eye was treated topically with the AR inhibitor CP-45,634 while the other served as control. The eye treated with CP-45,635 regenerated epithelium much more quickly than the untreated eye. Other AR inhibitors had similar beneficial effects.

INHIBITION OF LENS ALDOSE REDUCTASE BY FLAVONOIDS-THEIR POSSIBLE ROLE IN THE PREVENTION OF DIABETIC CATARACTS

Abstract Flavonoids, also referred to as vitamin P, were found to be highly potent inhibitors of aldose reductase, the enzyme that initiates cataract formation in diabetes. Over forty flavone derivatives were tested and found to be active but the two most potent ones were quercitrin and quercitrin 2″-acetate, which inhibit the enzyme activity by 50 per cent at 10 −7 and 4 × 10 −8 M respectively. The potency of these two compounds surpassed that of all the previosly known inhibitors of aldose reductase. Studies were conducted to determine how structural alteration in the basic flavonoid moiety affected their inhibitory ctivity. It is possible that further search may reveal even more potent analogues of this ubiquitously distributed group of plant polyphenols which may ultimately be useful in diabetic patients.

On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses

It has been suggested that the presence of high molecular weight protein aggregates in the lens can lead to light scattering and a consequent loss of transparency. We have measured the concentration of aggregates having a molecular weight greater than approximately 150 × 106 g/mole present in the soluble fraction of both aging normal and cataractous human lenses. This protein population is approximately 5% of the total soluble protein in lenses up to age 75 and increases to 10–15% in lenses aged greater than 75 years and in cataracts. The amino acid composition of the heavy molecular weight soluble aggregates of normal lenses is different in leucine content from alpha, beta and gamma crystallins and different in tyrosine content from beta and gamma crystallins. The aggregates from cataractous lenses show a greater increase in leucine and greater decrease in tyrosine. An unidentified component is present in the cataractous aggregates. Furthermore, calcium, an ion which increases in concentration in cataractogenesis, induces alpha crystallin to aggregate and induces the heavy molecular weight fraction to aggregate. This aggregation is irreversible by dialysis or chelation.

Aldose reductase and ϱ-crystallin belong to the same protein superfamily as aldehyde reductase

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH‐dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40–50% homology with ϱ‐crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and ϱ‐crystallin are members of a superfamily of related proteins.

The absence of cataracts in mice with congenital hyperglycemia.

Abstract 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.

Role of aldose reductase in the development of diabetes-associated complications

The aldose reductase-initiated intracellular accumulation of polyols has been clearly shown to have a hyperosmotic effect on the lens of experimental animals. Similar localized osmotic changes resulting in the onset of pathology may also occur in the nerve, corneal epithelium, and retinal pericytes. Except for experimental diabetic cataracts, however, the exact mechanism by which aldose reductase is involved in these diabetes-associated complications remains to be clarified.

Effects of lipid peroxidation products on the rat lens in organ culture: A possible mechanism of cataract initiation in retinal degenerative disease

Rat lenses in organ culture which are exposed to bovine rod outer segments (ROS) or to the major fatty acid of ROS, docosahexaenoic acid, are impaired in their ability to accumulate radiolabeled compounds which lenses normally accumulate by active processes. The extent of lens damage correlates well with the extent of lipid peroxidation in the culture medium as assessed by the thiobarbituric acid assay. Addition of vitamin E to the medium inhibits the effect on the lens while addition of Fe-ADP complexes potentiates the effect. Thus, the lens damage appears to be attributable to toxic species generated by peroxidation of the polyunsaturated lipid added to the culture medium. Toxic aldehyde products appear to be major mediators of the lens damage, since semi-carbazide, which avidly reacts with aldehydes, can protect lenses in this system. These findings may have relevance to the cataracts clinically associated with retinal degenerative diseases such as retinitis pigmentosa. The highly membranous photoreceptor cells are extremely rich in polyunsaturated lipid. Degeneration of these cells, which is the primary pathology in such diseases, would likely lead to peroxidation with generation of toxic products within the eye. Such products could potentially produce secondary damage to other ocular structures including the lens.

Philly mouse: A new model of hereditary cataract

Abstract Philly mouse is a new strain of mice derived from the Swiss-Webster strain which develops hereditary cataracts visible to the naked eye ca. 5–6 weeks after birth. Slit lamp examinations of the apparently clear lenses of 15 day mice indicate the presence of faint anterior opacities which progress, involving the suture area, by day 25. By 30 days a nuclear opacity develops which surrounds the nucleus by day 35. At the same time the anterior subcapsular opacity becomes diffuse and pronounced as the cataract becomes obvious to the naked eye. Biochemical studies indicate that an osmotic cataract is formed in the Philly mouse. By ca. 20 days of age there is an increase in lens water along with an alteration in electrolyte levels. Lenticular sodium rapidly increases while potassium levels decrease. Concomitant with cataract formation is an increase in total lenticular calcium and a decrease in lens dry weight, reduced GSH and ATP. In transport studies, no significant difference between the Philly and control lens was seen in the accumulation of AIB. When rubidium was substituted for potassium a decreased accumulation in the Philly lens older than 20 days was correlated with increased rubidium leak-out. This decreased accumulation due to increased leak-out appears to be the key biochemical change that accounts for osmotic cataract formation and it suggests the possibility of a defect in membrane permeability.

Structural changes in bovine lens crystallins induced by ascorbate, metal, and oxygen

Ascorbate, Fe3+, or Cu2+ and oxygen induced the oxidation of bovine lens crystallins. The modifications mimicked those that occur in the lens with aging. The modifications included the formation of nondisulfide crosslinks in alpha- and beta H-crystallin and the cleavage of alpha-, beta H-, and the low molecular weight crystallin fractions. In all three fractions, there was a loss of the more basic protein species and an increase in the more acidic species. Nontryptophan fluorescence with emission spectra between 400 and 500 nm was produced in beta H-crystallin. Cu2+ was less effective than Fe3+ in catalyzing the modification of beta H- and gamma-crystallin. Both metal ions were equally effective in catalyzing the modification of alpha-crystallin.

Aldose Reductase in the Diabetic Eye XLIII Edward Jackson Memorial Lecture

In diabetic cataracts aldose reductase initiates the cataractous process by converting glucose to sorbitol. The ensuing osmotic change, caused by sorbitol accumulation, adversely affects the lens permeability barrier so that the distribution within the lens of electrolytes, amino acids, and myo-inositol becomes grossly altered. These changes affect lens viability resulting in opacification. That aldose reductase triggers the process is shown by the fact that several structurally unrelated aldose reductase inhibitors prevent cataracts from occurring. Aldose reductase is also implicated in diabetic retinopathy and keratopathy. Aldose reductase functions in the retinal capillary pericytes, the cells first affected in microvascular abnormalities in diabetes. Additionally, retinal capillary basement membrane thickening can be prevented by aldose reductase inhibitors. Clinical trials are underway to determine the efficacy and safety of aldose reductase inhibitors in treatment of diabetic retinopathy.