Supplementing glucose metabolism in human senile cataracts

Transcription

1 Supplementing glucose metabolism in human senile cataracts Hong-Ming Cheng, Leo T. Chylack, Jr., and lngrid von Saltza Assay of the activities of hexokinase, phosphofructokinase, and pyruvate kinase showed that the first two declined in aging human lens cortex and all three enzymes retained constant activities in the epithelium throughout life. Moreover, both clear and cataractous aging lenses contained the same enzyme activities. ATP contents in cataracts, however, were lower than in clear lenses; in fact, after incubation at 37.5 C in isotonic (290 to 300 mosm), glucosecontaining media, ATP teas rapidly lost from cataracts (but not from clear lenses), suggesting excessive ATP expenditure in cataracts for osmotic balance. Cataracts incubated in media containing either glucose-6-phosphate or fructose-1,6-diphosphate produced significantly higher ATP than with glucose in the media, indicating that glucose metabolism in human senile cataracts could be supplemented with hexose phosphates. Fructose-1,6-diphosphate appeared to be more efficient than glucose-6-phosphate in preventing lens swelling during incubation. (INVEST OPHTHALMOL VIS SCI 21: , 1981.) Keywords: glycolysis, human senile cataracts, ATP, lactate, glucose-6-phosphate, fructose-l,6-diphosphate, supplementing glucose metabolism ippel,' after reviewing reports by several investigators, concluded that lens aging was indeed accompanied by a decline of glycolytic activity in the cytoplasmic compartment of the lens. To account for such a decline, numerous studies on the specific activities of glycolytic enzymes, the concentrations of glycolytic intermediates and cofactors, and the kinetic and biophysical properties of the From the Howe Laboratory of Ophthalmology, Harvard Medical School, and the Massachusetts Eye and Ear Infirmary, Boston, Mass. This project was supported by U.S.P.H.S. Grant EY01276 and, in part, by the Cooperative Cataract Research Group (CCRG) and by the Brigham Surgical Group Foundation, Brigham and Women's Hospital, Boston, Mass. Submitted for publication Oct. 9, Reprint requests: Dr. Hong-Ming Cheng, Howe Laboratory of Ophthalmology, 243 Charles St., Boston, Mass enzymes have been performed (for general reviews see Kuck 2 and Cotlier 3 ; for an exhaustive list of specific studies see Ohrloff 4 ). These studies indicate that in old lenses, there is a decrease of specific enzyme activities, with the outer layers of the lens retaining the highest enzyme activities (and metabolites, including ATP), and some enzymes seem to have undergone posttranslational alterations resulting in decreased affinities for their substrates. To be a ratelimiting factor of glycolysis in the aging lens, the enzyme must have either decreased to a critically low level or changed to a low catalytic state. Evidence for the latter has been presented by Hockwin, 5 who found accumulation of hexose monophosphates in old cattle lens and suggested that phosphofructokinase (PFK) had assumed the role of the primary regulator in the aging lens. Cheng and Chylack 6 confirmed this with a rat lens dispersion study, in which they found that /81/ $00.70/ Assoc. for Res. in Vis. and Ophthal., Inc.

2 Volume 21 Number 6 Glucose metabolism in human cataracts 813 the rate of lactate production from fructose- 6-phosphate dropped substantially in 8- to 12-month-old lens. Bous et al. 7 further substantiated the rate-limiting role of PFK in bovine lens by demonstrating that the enzyme affinity for fructose-6-phosphate declined 10-fold in old lenses. However, evidence for a critically low enzyme activity has never been found. It should be noted that most of the studies cited above were performed on animal lenses. It is doubtful as to whether the conclusions drawn from these studies can be extrapolated to human lens. For example, there are discrepancies in the properties of the three key enzymes of glycolysis between animal and human lenses. (1) Hockwin et al. 8 suggested that the high K ucose hexokinase (HK) isozyme in bovine lens was derived from a low j^ciucose f orm as the lens cells aged, whereas Chylack 9 found no difference in the proportion of the two forms between a 2.5-month-old infant lens and senile cataracts. (2) We found no accumulation of hexose monophosphate in the deep cortex of human aging lens (see Results), suggesting that PFK did not control glycolysis in this region. (3) Banroques et al. 10 found that there was a loss of allosteric pyruvate kinase (PK) isozymes in aging rabbit lens cells, whereas Cheng et al. 11 found that PK retained full allosteric property in human aging lens. A reassessment of the glycolytic status in the aging human lens is therefore necessary. The next question is how the low energy production in the aging lens contributes to cataractogenesis. One of the major expenditures of lens ATP is in the maintenance of osmotic balance, chiefly the ionic pump. Chylack 12 and Chylack and Schaefer 13 have shown that the lens lost its electrolyte balance when the energy source was deprived. We have observed a drastic increase of the wet weights when the human cataracts were incubated in isotonic (290 mosm) medium containing 5.5 mm glucose, whereas, in contrast, swelling of the clear lenses remained minimal. l4 This is in agreement with the general observation that cataracts are deficient in osmotic equilibrium due to loss of membrane integrity. l5 However, if the energy production is sufficient to satisfy the demand, there should be little or no swelling. The extensive swelling of incubated cataractous lenses in isotonic medium then suggests that ATP production in these lenses is inadequate. An examination of this possibility is therefore needed. Finally, encouraged by the discovery of a transport system for hexose phosphates in Escherichia coli 16 and the recent work by Korte et al., 17 who found that bovine lenses were permeable to fructose-1,6-diphosphate (FDP) that is, obviously not all biological membranes are impermeable to sugar phosphates we have decided to substitute sugar phosphates for glucose, hoping to bypass rate-limiting steps in glycolysis, e.g., HK and PFK, to see whether the ATP level can be elevated and lens swelling during incubation prevented. If this can be accomplished, osmotic stress exceeding the capacity of the membrane transport system can be alleviated. This report is a preliminary report of the three broad areas mentioned above. Materials and methods Human clear lenses were obtained from the New England Eye Bank and used within 24 hr after death. Human cataracts were collected in the operating rooms of the Massachusetts Eye and Ear Infirmary and classified according to Chylack 18 and were generally used within 2 hr after extraction. Only intact, unruptured lenses were used in all studies. The cataract classification data were stored in the Cooperative Cataract Research Group (CCRG) Computer Data Bank and are available on request. Data in this report did not consist of comparative studies of cataract types; lens classification information was therefore omitted. Male Sprague-Dawley albino rats were purchased from Charles River Breeding Laboratories, Wilmington, Mass. Assay procedures for HK, PFK, and PK have been described previously by Chylack et al., 19 Cheng and Chylack, 20 and Cheng et al." To examine the topographical distribution of these enzymes in human lens, the capsule-epithelium with adhering superficial cortex was first removed, and

3 814 Cheng et al. Invest. Ophthalmol. Vis. Sci. December 1981 Table I. Total enzyme activity in human lens Lens type* Clear, juvenile HK C PFK PK Clear, senile HK C PFK Enzyme activity (nmol/min /tissue, mean ± S.D.) Epithelium PK Cataractous, senile HK C PFK PK ± 0.41 ± ± ± 1.40 ± ± ± 0.65 ± 8.95 ± Cortex ± ± ± dt dt d: dt d: d: A Clear, juvenile lenses were 4-11 years of age, 4 lenses; senile lenses were more than 55 years old, 4 clear lenses and 12 cataracts. B Loss of cortical HK in senile lenses was significant to p<0.1 for clear lenses and p<0.01 for cataractous lenses; loss of cortical PFK was significant to p<0.01 for both types of senile lenses vs. juvenile lenses. c Soluble hexokinase only. Total (soluble + insoluble) hexokinase activity was 3.71 ± 1.41 (4 lenses), and 3.72 ± 1.60 (24) in the epithelium of clear and cataractous senile lenses, respectively. the deep cortical and the nuclear fractions were separated with a cork borer (5 mm ID). These lens fractions were then homogenized in 0.05M Tris Cl buffer (ph 7.4) and centrifuged at 27,000 x g at 0 to 4 C in a Sorvall refrigerated centrifuge for 15 min to obtain the soluble fraction; this procedure allowed total recovery of PFK. For short-term (6 hr or less) whole-lens incubating studies, Dulbecco's phosphate-buffered saline (Microbiological Associates, Bethesda, Md.) was used as the basal medium, to which the substrates (5.5 mm each) were added; the osmolarity of the final mixture was kept at 300 mosm. For overnight incubating studies, the TC199/bicarbonate medium with each substrate at 12 mm was used, with the osmolarity maintained at 295 ± 5mOsm. 12 The lenses were incubated in 12 ml of medium at 37.5 C in a humid CO 2 chamber. There was no significant difference in the rates of lactate production with the lenses incubated in either medium. To assay for glycolytic intermediates such as glucose-6-phosphate (G6P), fructose-6-phosphate, FDP, and triose phosphates, the incubated lenses were washed five times, each time with 100 ml of normal saline (kept at 37.5 C) to avoid contaminations from the incubating media. Lens fractions were quickly frozen with liquid N 2 and homogenized in 0.5 ml of 6% perchloric acid. The homogenates were centrifuged, and the supernatants were neutralized with 5M K 2 CO 3 solution and centrifuged again. The final supernatants were assayed enzymatically according to the method of Bergmeyer. 21 The rate of lactate production was determined on aliquots, periodically withdrawn from the incubating media, with Boehringer-Mannheim lactate assay kits. ATP contents in the lens were assayed according to the method of Strehler and Totter. 22 Lenses incubated in the presence of ADP were rinsed thoroughly with warm normal saline to eliminate contamination. To avoid interference from myokinase (which converted ADP to ATP) present in the firefly lantern/luciferase preparations, the aliquots for ATP assays were limited to a volume that contained a maximal ADP of 1 nmol. However, prolonged (18 to 24 hr) incubation of lens in ADP tended to result in higher background level of ATP even with the use of myokinase-free luciferase. Lens wet weights could be determined only after incubation, since abrasion, mechanical damage, and ruptures during drying and weighing invariably occurred, causing wide fluctuations of the measured values when incubation after weight determination was attempted. All statistical analyses were unpaired Student t tests. All auxiliary enzymes and biochemicals were products of Boehringer-Mannheim, Indianapolis, Ind. Firefly lanterns and myokinase-free luciferase were obtained from Sigma Chemical Co., St. Louis, Mo. Cryoprobe for human lens extraction was a product of Sparta Instrument Corp., Fairfield, N. J. Results Glycolytic status in human lens. In surveying the activities of HK, PFK, and PK in human lenses, we found an age-related decline of both HK and PFK only in the cortical region of the aging lens (Table I). Epithelial enzyme activities, on the other hand, remained relatively constant throughout life. An assay on the glycolytic intermediates showed that there was no detectable G6P or fructose-6-phosphate (assayed on six freshly extracted cataracts) in the cortex, suggesting that (1) the feed-back inhibition of HK did not operate, (2) PFK was not rate-limiting,

4 Volume 21 Number 6 Glucose metabolism in human cataracts 815 Table II. ATP content of aging human lenses (more than 55 years old) incubated in glucose-containing media with osmolarity adjusted to mosm ATP content (fxmol/gm of lens) Incubation time (hr) Clear lens p* Cataracts p* ± 0.27(12) 1.26 ± 0.29(3) 1.12 ± 0.19(4) 1.12 ± 0.56(28) 0.53 ± 0.20(4) < ± 0.39(6) < ± 0.20(5) <0.05 <0.01 Values shown are mean ± S.D.; number oflenses in parentheses. ""Comparisons with zero-time values. Table III. Glycolytic intermediates (nmol/tissue) in lens tissues incubated in Dulbecco's phosphate-buffered saline containing 5.5 mm glucose or FDP Epithelium Cortex Intermediates With glucose With FDP With glucose With FDP G6P F6P FDP TPs 0.47 ± 0.55(11) 1.35 ± 0.88* 1.17 ± 0.70t 0.09 ± 0.21(13) 6.49 ± 4.01* 4.30 ± 2.78t (5) (5) 2.51 ± ± ± ± 2.03 F6P = fructose-6-phosphate; TPs = triose phosphates; = non-detectable. Values are mean ± S.D.; number oflenses in parentheses. *p < 0.001, vs. glucose- vs. FDP-incubated lenses, tp < 0.01, glucose- vs. FDP-incubated lenses. and (3) residual HK activity alone regulated glycolysis in the aging cortex. Table I also shows that there was no difference between clear and cataractous aging lenses in terms of enzyme activities, suggesting that the loss of cortical HK and PFK is part of the aging process but is apparently not related to cataractogenesis directly. In addition, an assay of the initial rates of lactate production showed that clear and cataractous lenses produced 0.48 and 0.62 /u-mol of lactate per hour per lens, respectively (data based on two clear lenses and four cataracts). Since the glycolytic machinery is identical in both clear aging and cataractous lenses, ATP production should also remain the same. However, ATP contents in senile cataracts were slightly lower (significant to p < 0.1) than those of the clear lenses (Table II, zero-time values). Since clear lenses were unavoidably delayed for up to 24 hr (stored at 0 to 4 C in intact eyes) before assayed for ATP, the data may represent an underestimate. (In fact, rat lenses in intact eye globes stored at 0 to 4 C for 24 hr lost up to 37% of ATP as compared to unstored, fresh lenses.) The demand for ATP is probably much greater for cataracts in vivo; when intact aging lenses were incubated in isotonic (290 to 300 mosm) media (see Materials and methods), the lenses tended to swell, 14 but ATP contents in cataracts rapidly declined at a rate much faster than that of clear lenses (Table II), suggesting that the production of ATP in cataractous lenses could not keep pace with the consumption. Such a difference was probably not due to surgical trauma to the cataracts, since a pair of clear lenses, with one extracted with a cryoprobe, showed similar ATP contents (1.27 vs /umol/gm of lens, with the latter cryoprobed) after being incubated for 24 hr in TC199/bicarbonate containing 12 mm glucose. Furthermore, the ATP decline was not due to ATP leak-out from the incubated cataracts; no ATP was detected in the incubating media (tested on

5 816 Cheng et al. Invest. Ophthalmol. Vis. Sci. December 1981 Table IV. ATP contents of incubated cataracts Incubation time (hr) Addition Glucose G6P FDP Glucose/FDP None Glucose/ADP G6P/ADP FDP/ADP ADP Glucose/ADP G6P/ADP FDP/ADP ADP ATP (ixmole/ gin of lens) 0.67 ± 0.39(6) 0.37 ± 0.24(6) 0.68 ± 0.33(10) 0.66 ± 0.43(8) 0.47 ± 0.23(6)* 0.67 ± 0.31(6) 0.90 ± 0.09(6) 1.07 ± 0.33(15) 0.57 ± 0.32(7)* 1.15 ± 0.23(6) 1.60 ± 0.62(8) 2.23 ± 0.94(7) 0.98 ± 0.28(7)* P <0.4 <0.5 <0.2 <0.4 <0.6 <0.05 <0.01 <0.3 <0.05 <0.01 Values are mean ± S.D.; number of lenses in parentheses. * Background values for statistical comparisons. four lenses incubated in Dulbecco's saline for 6 hr). Alternative substrates for lens glycolysis. In order to increase ATP supply in the aging lens, rate-limiting and ATP-consuming steps such as HK and PFK must be bypassed. Two glycolytic intermediates, i.e., G6P and FDP, seem promising; the latter also activates PK 11 and would appear to be a better substrate. Penetration of the substrates. Human cataracts were incubated in Dulbecco's phosphate-buffered saline containing 5.5 mm glucose, G6P, or FDP. After 3 hr at 37.5 C, the lenses were vigorously washed (see Materials and methods) and assayed for a number of glycolytic intermediates. G6P did not accumulate in the lens (results not shown). In the presence of FDP, however, there was significant increase of both FDP and triose phosphates in the epithelium + superficial cortical fraction (Table III). The disparity between G6P and FDP indicates that there was no constant intercellular pool for hexose phosphates in the cataractous lens and probably reflects the difference in the rate of diffusion and/or utilization of the two substrates. Utilization of the substrates. Lenses were incubated for a minimum of 6 hr. This time period was chosen because these lenses produced lactate linearly for 3 hr at 37.5 C with G6P, FDP, or ADP or without any substrate in the medium before the production ceased; presumably the lenses were capable of using endogenous substrates during the first 3 hr of incubation. However, these lenses lost at least 40% of their ATP after 6 hr of incubation (Table IV). Our previous studies with rat lens dispersions indicated that the availability of ADP was a limiting factor in glycolysis. 6 ADP was therefore added to the incubating medium. In the presence of ADP, ATP contents were significantly elevated with either G6P or FDP as the substrate (Table IV). Supply of ADP through endogenous sources, i.e., kinase reactions, appeared inadequate, since lenses incubated with both glucose and FDP failed to show increase of ATP (Table IV, 6 hr incubation without ADP). Prolonged (18 to 24 hr) incubation also showed that G6P and FDP generated significantly higher ATP in the presence of ADP than the background control (Table IV). The difference in ATP production from G6P and FDP can be attributed to the consumption of 1 mol of ATP for the phosphorylation of each mole of G6P, but not needed for FDP utilization. It should be noted, however, that human clear aging lenses incubated in FDP + ADP failed to show higher ATP contents than those incubated in glucose alone (1.26 ± 0.29 /u-mol/gm of lens with glucose as the substrate vs ± 0.29 with FDP + ADP; incubation 6 hr at 37.5 C; determined on three lens pairs); however, lenses incubated without any substrate showed a 50% loss of ATP, indicating that FDP + ADP was utilized but was probably limited by the high ATP contents (which inhibited PK 11 ) in these lenses (Table II) and/or that these lenses were relatively impermeable to FDP and ADP (see Discussion). Wet weights. The wet weights of lenses incubated for 24 hr in the presence of ADP, glucose/adp, G6P/ADP and FDP/ADP were ± 28.1 mg (seven lenses), ± 27.0

6 Volume 21 Number 6 Glucose metabolism in human cataracts 817 (five lenses), ± 28.9 (seven lenses), and ± 30.5 (eight lenses), respectively. Based on a population mean of ± 30.6 mg (40 lenses) the FDP/ADP-incubated lenses showed the least amount of increase: 9.11% (p < 0.1); G6P/ADP-, glucose/adp-, and ADP-incubated lenses increased 11.6% (p < 0.05), 12.2% (p < 0.05), and 14.2% (p < 0.01), respectively. This suggests that FDP is effective in preventing lens swelling. Discussion It should be noted that although the loss of HK in the cortex of the aging human lens is quite extensive, the epithelial fraction retains the same glycolytic enzyme activities as those in juvenile lenses (Table I), suggesting localization of glycolysis in this fraction. This is in agreement with Sippel's conclusion 1 that glycolytic activities in the outer layers of the lens remains constant. Loss of cortical HK possibly decreased the overall rate of glycolysis; this is evidenced by the virtual absence of hexose monophosphates from the cortices (Table III). The lack of hexose monophosphates cannot be attributed to loss through defective lens membrane in cataracts, for the rates of lactate production are essentially the same for clear and cataractous lenses. Since glycolytic machinery in clear and cataractous lenses is identical, it is necessary to conclude that disintegration of lens membrane is independent from low glycolytic activity; the latter simply amplifies the consequence of the former (i.e., electrolyte imbalance), by failing to provide excessive usable ATP (Table II) for the ionic pump. It is encouraging to find that in the presence of ADP, both G6P and FDP are able to increase lens ATP (Table IV) and that FDP is effective in limiting lens hydration (see Results). The permeability of FDP does not seem to be specific to cataractous lens membrane. Korte et al. 17 have succeeded in promoting the FDP level in intact bovine lenses (they also demonstrated that rabbit corneas were permeable to FDP). However, these lenses, like human clear lenses, failed to show elevated ATP levels, and ATP remained at physiologic concentration when the lenses were incubated with FDP. Apparently these lenses, also like human clear lenses, do not expend ATP as rapidly as human senile cataracts (Table II). The therapeutic significance of hexose phosphate treatment in preventing the progression of cataract formation is now under evaluation. It appears that osmotic stress in lenses with minimal membrane damage can be eliminated with such a treatment. REFERENCES 1. Sippel TP: Energy metabolism in the lens during aging. INVEST OPHTHALMOL 4:502, Kuck JFR Jr: Metabolism of the lens. In Cataract and Abnormalities of the Lens, Bellows JG, editor. New York, 1975, Grune & Stratton, Inc., p Cotlier E: The lens. In Adler's Physiology of the Eye. Moses RA, editor. St. Louis, 1981, The C. V. Mosby Co., p Ohrloff C: Age changes of enzyme properties in crystalline lens. In Lens Aging and Development of Senile Cataracts, Hockwin O, editor. Basel, Switzerland, 1978, S. Kager (Interdisciplinary Topics in Gerontology, vol. 12, p. 158). 5. Hockwin O: Enzyme activities in relationship to age and phosphorylation intermediates in energy metabolism. INVEST OPHTHALMOL 4:496, Cheng HM and Chylack LT Jr: Factors affecting the rate of lactate production in rat lens. Ophthalmic Res 9:381, Bous F, Hockwin O, Ohrloff C, and Bours J: Investigations on phosphofructokinase (PFK, E.C ) in bovine lenses in dependence on age, topographic distribution and water soluble protein fractions. Exp Eye Res 24:383, Hockwin O, Ohrloff C, and Jeschke G: Hexokinase activity of bovine lenses. Presented at Annual Meeting of the Association for Research in Vision and Ophthalmology, Chylack LT Jr: Human lens hexokinase. Exp Eye Res 15:225, Banroques J, Gregori C, and Schapira F: Aging of pyruvate kinase isozymes in rabbit lens. Exp Eye Res 27:427, Cheng HM, Chylack LT Jr, and Chien J: Control of pyruvate kinase activity in the lens. Exp Eye Res 27:39, Chylack LT Jr: Mechanism of "hypoglycemic" cataract formation in the rat lens. I. The role of hexokinase instability. INVEST OPHTHALMOL 14:746, Chylack LT Jr and Schaefer FL: Mechanism of "hypoglycemic" cataract formation in the rat lens.

7 818 Cheng et al. Invest. Ophthalmol. Vis. Sci. December 1981 II. Further studies on the role of hexokinase instability. INVEST OPHTHALMOL 15:519, Chylack LT Jr, Henriques HF, III, and Tung WH: Inhibition of sorbitol production in human lenses by an aldose reductase inhibitor. Doc Ophthalmol Proc Ser 18:65, Kuck JFR Jr: Composition of the lens. In Cataract and Abnormalities of the Lens, Bellows JG, editor. New York, 1975, Grime & Stratton, Inc., p Dietz GW, Jr: The hexose phosphate transport system of Escherichia coli. Adv Enzymol 44:237, Korte I, Hockwin O, and Kaskel D: Utilization of fructose-1,6-diphosphate as glycolytic substrate in bovine lens homogenates. Doc Ophthalmol Proc Ser 18:163, Chylack LT Jr: Classification of human cataracts. Arch Ophthalmol 93:888, Chylack LT Jr, Kinoshita JH, and Kasabian R: Nature and distribution of two distinct forms of hexokinase within the mammalian lens. Exp Eye Res 10:250, Cheng HM and Chylack LT Jr: Properties of lens phosphofructokinase. INVEST OPHTHALMOL 15:279, Bergmeyer HU: Methods of Enzymatic Analysis. New York, 1974, Academic Press, Inc., vol. Ill, pp and Strehler BL and Totter JR: Determination of ATP and related compounds. Firefly luminescence and other methods. Methods Biochem Anal 1:341, 1954.