Metabolic pathways in experimental diabetic cataract. Sidney Lerman

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1 Metabolic pathways in experimental diabetic cataract Sidney Lerman Specific areas in carbohydrate and protein metabolism were studied in the lenses of alloxandiabetic rats. The studies were performed at weekly intervals commencing with the first week after diabetes had been induced and continuing until the fourth to fifth week. The results of these investigations indicate that lenticular anaerobic glycolysis as reflected by lactate production and the recovery of C--O t from lenses incubated with glucose--c-, remains relatively unchanged during the to 5 week period compared with that from control animals. The concentration of glucose and fructose in the diabetic lenses shows a fifteen- to twentyfold increase one week after diabetes has been induced and remains at this elevated level throughout the experimental period. The recovery of C--O s from lenses incubated with glucose-l-c- shows a marked decline at the first week (in contrast with the recovery of C--O : from glucose--c-) and remains low in the diabetic lenses throughout the to 5 week period of observation. The level of ATP in the lenses also exhibits a similar decrease of approximately 35 to per cent. In studying the incorporation of C--labeled leucine, lysine, and valine, a marked decline in the degree of incorporation of these amino acids into lenticular protein quickly becomes manifest in the diabetic lenses. There is a similar decrease in the free amino acid concentration in the lenses which becomes apparent within the first week of diabetes. In view of the large number of papers that has accumulated in the literature on various aspects of experimental diabetic cataractogenesis, only those papers which in the author's opinion are pertinent to this report will be cited. A more detailed bibliography may be obtained from a review on cataract formation in human and experimental diabetes which was published two years ago. In an attempt to define some of the From the Department of Surgery, Division of Ophthalmology, and the Department of Biochemistry of the University of Rochester School of Medicine and Dentistry, Rochester, N. Y. This work was supported by United States Public Health Service Grant B-38 and in part by research funds granted by The Rochester Eye- Bank and Research Society, Inc. 57 metabolic alterations which may develop in the lenses of alloxan-diabetic rats, specific areas in carbohydrate and protein metabolism were investigated in these organs. Materials and methods Holtzman strain white male rats weeks of age and weighing 7 to 8 grams were employed in all the. Diabetes was induced by starving the animals for 8 hours, then giving an injection of one-fourth unit of insulin, and, to 5 hours later, mg. per kilogram alloxan. Blood sugar determinations were performed to 3 days after the injection of alloxan and were repeated days later. Only those animals in which the blood sugar level remained above 3 mg. per cent were employed in these studies. The control and experimental animals were maintained on a standard diet throughout the experimental period. A specific number of control and alloxan-diabetic rats were sacrificed at weekly intervals as shown in the accompanying tables. The variation in the number of rats used per experiment was based on the number of survivors at that time period. Downloaded From: on 7//8

2 58 Lerman Investigative Ophthalmology August 9 Table I. Concentration of lactate, glucose, and fructose in the normal and alloxan-diabetic rat lens Average lens weight (mg.). 3.3 Lactate (tig/lens). 9.8 Glucose (fig/lens). 8.9 Fructose (vg/lens).7. weeks Table II. The recovery of C--O from lenses incubated with glucose-l-c- or glucose--c- Average lens weight (mg.) C--Os recovered from glucose-l- C- c.p.m./gm. sol. protein,9,8 C--Os recovered from glucose-- C- c.p.m./gm. sol. protein weeks ,83, ,33,59,57, The rats employed in the amino acid incorporation studies were starved for to hours prior to the intraperitoneal injection of the specific C- amino acids. A total of 5.5 /*c of either C-- leucine, lysine, valine, or aspartic acid was injected into each rat and the animals were sacrificed 3 hours later. In those involving the nonmetabolizable amino acid (C- alpha aminoisobutyric acid), a similar amount was injected. Adenosine triphosphate (ATP) determinations were performed according to the method of Strehler and Totter, lactate by the method of Barker and Summerson, 3 fructose by Dische and Borenfreund's method, and glucose by the glucose oxidase method. 5 The methods utilized in performing the C- amino acid incorporation studies and the incubation studies with glucose-l-c- or glucose--c- have been described in previous communications. ' 7 The following procedure was employed to obtain a protein-free filtrate from the lens homogenates. The insoluble protein was removed by centrifugation at g for minutes. A small amount of per cent trichloracetic acid (TCA) was added to the supernatant to obtain a 5 per cent TCA solution. This was heated at 9 C. for 5 minutes and then centrifuged at 3, r.p.m. for 5 minutes. The precipitate was retained Downloaded From: on 7//8

3 Volume Number Metabolic pathways in experimental diabetic cataract 59 for counting and for nitrogen analysis by the micro-kjeldahl method. The supernatant which contained the free amino acid fraction as well as other acid soluble constituents, e.g., nucleotides, was retained for chromatography and counting. Results The results of the lactate, glucose, and fructose determinations in the lenses from the normal and alloxan-diabetic rats are shown in Table I. The concentration of lactic acid per lens was essentially the same in the alloxan-diabetic rats as compared with the control animals throughout the entire period under observation. However, there was a marked increase in the glucose levels in the diabetic rat lens which was apparent one week after the injection of alloxan, and it remained elevated thereafter. The concentration of fructose in the lenses showed a similar and even more marked elevation. The recovery of C--O L. from lenses incubated with glucose-l-c- or glucose- -C- is shown in Table II. In the oneweek diabetic lenses the recovery of C--O from glucose-l-c- fell to considerably less than half the corresponding amount obtained from the control lenses. This marked fall in C--O-. recovery from the diabetic lenses continued throughout Table III. The concentration of ATP in the normal and alloxan-diabetic rat lens Average lens weight (mg.) ATP (tig/lens) determinations 8 weeks Table IV. The incorporation of C- lysine and aspartic acid into the soluble protein fraction of the nonnal and alloxan-diabetic rat lens Lysine 3,8,9 Aspartic acid 7,397 5,98 Free lysine 78,59,583 weeks 3,988 7,93 3 3,587 3,3 3,88 5,998 8,79 3,99 9,988,398,998,599 Downloaded From: on 7//8

4 5 Lerman Investigative Ophthalmology August 9 the week experimental period. However, there was no significant difference in the recovery of C--O from glucose--c- between the diabetic and control lenses at any time. The concentration of ATP in the control and diabetic lenses is shown in Table III. In the alloxan-diabetic lens, the level of ATP fell by approximately one third after the first week of alloxan diabetes, and remained at this level for the next. The results of the in vivo C- amino acid incorporation studies are shown in Tables IV, V, and VI. In the diabetic lens, the degree of incorporation of C- lysine is almost 5 per cent lower than in the control lens during the first weeks of experimental diabetes. During the third and fourth weeks, the incorporation of C- lysine into the soluble protein fraction of the diabetic lens is even lower (approximately one sixth of the amount taken up by the control lens). The activity of C- lysine recovered in the free amino acid fractions of the lenses was also considerably lower in the diabetic lenses as compared with the control lenses. In both control and diabetic lenses, incorporation of C- aspartic acid was much lower as compared with the other amino acids employed. However, the diabetic lens also showed a marked decline in the amount of C- aspartic acid incorporated at the first and fourth weeks of diabetes as compared with the control lens (Table IV). There was also a marked fall in the degree of incorporation of the two neutral amino acids employed (C- leucine and valine) in the diabetic lenses (Table V). The decline was a progressive one; from to 5 per cent at the first week to about 7 per cent by the fourth week. The concentration of C- valine in the free amino acid fraction of the lens also showed a marked decrease in the diabetic rat. Table VI shows the results of the experiment in which C- alpha aminoisobutyric acid was employed. Since this is a nonmetabolizable amino acid, there was no incorporation into the soluble lens protein fraction. However, the concentration of this amino acid in the free amino acid fraction of these lenses again reveals that in the diabetic rat there is a very marked decrease in the level of this amino acid. Although an attempt was made to measure the concentration of the amino acids (lysine, aspartic, leucine, valine, and alpha aminoisobutyric) in the rat aqueous humor, the results were inconclusive. This was because of the difficulty encountered in obtaining constant amounts of aqueous humor from these animals without damaging the lens during the procedure. Discussion The marked elevation in the concentration of glucose in the lens of the alloxandiabetic rat reported upon here is in close agreement with the results obtained by Kuck. s Kuck proposed a transhydrogenase system involving the sorbitol pathway as depicted in the following reactions: Glucose + TPNH Sorbitol + DPN Sorbitol + TPN Fructose + DPNH Net Glucose + TPNH + DPN -* Fructose + TPN + DPNH He suggested that such a transhydrogenase system coupled with the reduction of pyruvate and the shunt may serve as an indirect source of high energy phosphate in lenticular metabolism. Although such a system may be present in the normal lens, it is difficult to see how it could be a source for a significant amount of ATP. It is even more difficult to attribute the enhancement of the above sorbitol pathway (which occurs in the diabetic lens) as an important factor in diabetic cataractogenesis. If this pathway serves as a minor source of ATP in the normal lens, a marked enhancement of this pathway should be accompanied by an increase in ATP. One might also expect an increase in the concentration of lactate in such lenses since such a transhydrogenase system is apparently coupled with a reduction of pyruvate. There is little, if any, evidence that glycolysis is impaired in the early stages of alloxan-diabetic cataractogenesis. The results of our studies Downloaded From: on 7//8

5 CO CO Volume Number Metabolic pathways in experimental diabetic cataract 5 Table V. The incorporation of C- leucine and valine into the soluble protein fraction of the normal and diabetic rat lens Leucine 5,977 37,85 Valine 7,37 3,87 Free valine 5,,597 toeeks 77,59 9,99 to to 7,3 7,7 5,579 7,93 3,35 9,9 5,8 9,9 8 Table VI. The incorporation of C- alpha aminoisobutyric acid into the lens of the normal and alloxan-diabetic rat weeks c.p.m./gm. sol. protein Free alpha aminoisobutyric acid 7,97 7,397 to to 39,98 7,997 3 show that the concentration of lactic acid and the recovery of C--O from glucose- -C- remains essentially normal in such lenses. However, the concentration of ATP falls by approximately 35 per cent, and the recovery of C--O from lenses incubated with glucose-l-c- shows an even greater decline. The latter data are in agreement with other reports which indicate that the activity of the hexose monophosphate pathway is significantly decreased in the liver of alloxan-diabetic rats. "" Since there is no evidence of an increase in ATPase activity in the diabetic lens, one can at present account for the fall in the concentration of ATP in such lenses only on the basis of an inhibition of glycolysis, the Krebs cycle, or the hexose monophosphate shunt. The only pathway of glucose oxidation that appears to be affected in the alloxan-diabetic lens is the latter pathway. In view of the recent reports by Fitch and associates ' 3 it is not unlikely that the inhibition of G--P and -P-G dehydrogenase (and consequently of the hexose monophosphate shunt) that occurs in the liver and lens of alloxan-diabetic rats is related to a decrease in the use of that specific pathway. Thus, in the diabetic lens, the decrease in available giucose-- phosphate may be reflected by an inhibition of the direct oxidative pathway, whereas the glycolytic pathway utilizes whatever G--P is available. Since glycolysis is the more important pathway in the lens with respect to glucose metabolism, it is not inconceivable that such a mechanism might prevail. Downloaded From: on 7//8

5 Lerman Investigative Ophthalmology August 9 The marked increase in the level of glucose in the diabetic lens is apparently directly related to the concentration of glucose in the blood plasma and

6 5 Lerman Investigative Ophthalmology August 9 The marked increase in the level of glucose in the diabetic lens is apparently directly related to the concentration of glucose in the blood plasma and aqueous humor. s The result of this marked accumulation of glucose within the lens is an apparent stimulation of the so-called sorbitol pathway with a consequent marked increase in the level of this sugar alcohol in the diabetic lens. Kinoshita 5 has pointed out recently that in all three forms of sugar cataract (diabetic, galactose, and xylose) there is a marked accumulation of the corresponding sugar alcohol in the lens. Since the lens capsule is not permeable to these compounds, he suggested that the high levels of these sugar alcohols within the lens will be accompanied by a marked hypertonicity within the lens and a consequent imbibition of water. The hydropic degeneration of lens fibers which is a relatively early occurrence in all three forms of sugar cataract would thus be explained on this basis. Although the hypothesis may prove to be valid, it is difficult to conceive of it as a primary factor in the pathogenesis of the sugar cataracts. Severe disturbances in the basic metabolism of such lenses have been noted by many workers, and it would seem more likely that these factors would exert a more profound effect on the lens. The results of the amino acid incorporation indicate that there is a marked inhibition of the transport of the amino acids into the alloxan-diabetic lens. Although similar results could be obtained if the site of this inhibition were at the level of the blood-aqueous barrier, several aqueous taps indicated that the level of alpha aminoisobutyric acid in the aqueous of the diabetic rat is not sufficiently lower than that of the normal rat to fully account for the results if the other amino acids behave similarly. More definitive studies on the rabbit eye by Kinsey and Reddy G indicate that the transport of amino acids into both the aqueous humor and the lens of the diabetic animal is markedly inhibited. Preliminary studies in our laboratory indicate that this inhibition can be reversed by insulin. REFERENCES. Chodos, J. B., and Habegger-Chodos, H. E.: Cataract formation in human and experimental diabetes, Survey of Ophth. 5: 9 and, 9.. Strehler, B. L., and Totter, J. R.: Firefly luminescence in the study of energy transfer mechanisms. I. Substrate and enzyme determination, Arch. Biochem. & Biophys. : 8, Barker, S. B., and Summerson, W. H.: The colorimetric determination of lactic acid in biological materials, J. Biol. Chem. 38: 535, 9.. Dische, Z., and Borenfreund, E.: A new spectrophotometric method for the detection and determination of keto sugars and trioses, J. Biol. Chem. 9: 583, Glucostat reagent procedure, Worthington Biochemical Corporation, Freehold, N. J.. Lerman, S., Devi, A., and Hawes, S.: Incorporation of labelled amino acids into lens protein of normal, glactose and xylosefed rats, Am. J. Ophth. 5: (Pt. II), Lerman, S.: Pathogenetic factors in experimental galactose cataract, A. M. A. Arch. Ophth. 3: (Pt. II) 3, Kuck, J., Jr.: The formation of fructose in the ocular lens, A. M. A. Arch. Ophth. 5: 8, Bloom, B., Eisenberg, F., Jr., and Stetten, D., Jr.: Glucose catabolism in liver slices via the phosphogluconate oxidation pathway, J. Biol. Chem. 5:, Felts, J. M., Doell, R. G., and Chaikoff, I. L.: The effect of insulin on the pathways of conversion of glucose to fatty acids in the liver, J. Biol. Chem. 9: 73, 95.. Glock, G., McLean, P., and Whitehead, J. K.: Pathways of glucose catabolism in rat liver in alloxan diabetes and hyperthyroidism, Biochem. J. 3: 5, 95.. Fitch, VV. M., Hill, R., and Chaikoff, I. L.: Hepatic glycolytic enzyme activities in alloxandiabetic rat: Response to glucose and fructose feeding, J. Biol. Chem. 3: 8, Fitch, W. M., and Chaikoff, I. L.: Extent and patterns of adaptation of enzyme activities in livers of normal rats fed diets high in glucose and fructose, J. Biol. Chem. 35: 55, 9.. Van Heyningen, R.: Formation of polyols by the lens of the rat with sugar cataract, Nature 8: 9, Kinoshita, J.: Communication.. Kinsey, V. E., and Reddy, D. V. N.: Personal communication. Downloaded From: on 7//8

R/adiation cataractogenesis has been

R/adiation cataractogenesis has been The effect of ionizing radiation on protein and carbohydrate metabolism in the rat lens Sidney Lerman, Anima Devi, and Renaka Banerjee The effects of ionizing radiation on the rat lens were investigated