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The effect of high glucose and oxidative stress on lens metabolism, aldose reductase, and senile cataractogenesis
The Effect of High Glucose and Oxidative Stress on Lens Metabolism, Aldose Reductase, and Senile Cataractogenesis Hong-Ming
Cheng and R. Gilbert0
Gonzilez
Diabetic cataractogenesis, a multifactorial process, was examined with nuclear magnetic resonance (NMR). P-31 NMR spectroscopic studies showed substantial alteration of both energy and membrane metabolism in the diabetic lens. Findings from a C-13 NMR spectroscopic determination of the sorbitol pathway flux in lenses incubated in 35.5 mmol/L glucose revealed that (1) one-third of total glucose consumed was channeled through this pathway, and (2) the turnover rate of NADPH to NADP was 3,00O%/hr. Furthermore, a competition for NADPH between aldose reductase and glutathione reductase was demonstrated. It is important to note that all metabolic changes in hyperglycemic/diabetic lenses can be prevented by aldose reductase inhibitors, o 1986 by Grune & Stratton, inc.
eg, sorbinil.
T
HE INVOLVEMENT of the sorbitol pathway in diabetic cataractogenesis in animal models has long been established.‘-’ However, its role in diabetic human cataract formation is not as clear-cut. Sorbitol contents in postmortem diabetic human lenses, as well as in lenses incubated in high glucose, are much lower than those in experimental diabetic cataract models.“’ Based on aldose reductase kinetics, it was estimated that human lenses can generate 7 hmol sorbitol/lens/d at a glucose concentration of 35.5 mmol/L.’ This is, however, the maximal rate; in vivo production may be considerably less. Nonetheless. the osmotic stress theory may still apply if one argues that sorbitol accumulates in the epithelium and superficial cortex, where most aldose reductase activity resides. 8*9This is conceivable but difficult to verify. The osmotic stress issue is further complicated by extralenticular factors: (1) a concurrent increase of osmolarity in the aqueous and vitreous humors may offset the increase in lens tonicity; on the other hand, one can also argue that (2) the increase in aqueous glucose per se may be osmotically stressful to the lens,” particularly if the lens is incapable of compensating rapid fluctuation of glucose levels in the aqueous humor.” In tissues such as the peripheral nerve, kidney, and human lens that do not produce and accumulate large quantities of sorbitol, factors other than or in addition to the osmotic stress may also play a pathogenic role. In the animal lens model, these factors may have been masked by the acute osmotic imbalance caused by accumulation of lens sorbitol. We will examine several of these factors. PHOSPHORUS
METABOLISM
Activation of the sorbitol pathway is not an isolated event, ie, other interacting metabolic activities are also involved From the Howe Laboratory of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, Boston. Mass, and the Francis Biiter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Mass. Presented at the Pfizer Sorbinil Symposium-The Eflects of Sorbinil on the Pathophysiology of Diabetic Complications, Dorado, Puerto Rico, May 30-June 2, 1985. Supported by Grant EY04424 from the National Eye Institute. Address reprint requests to Hong-Ming Cheng, PhD, Howe Laboratory of Ophthalmology, 243 Charles St, Boston, MA 02114. o 1986 by Grune & Stratton, Inc. 0026-a495/86/3504-1003$03.00/O 10
(Fig 1). The interactions are mediated through cofactors and adenylates.” Stimulation of the hexose monophosphate shunt (HMPS) in conjunction with sorbitol pathway activation is well known.13 Other activities are less obvious but have become apparent after lens metabolism is evaluated with P-3 1 nuclear magnetic resonance (NMR) spectroscopy, which provides information on all phosphorus metabolites above 0.2 mmol/L in concentration (with present-day technology). P-31 NMR studies showed that activation of the sorbitol pathway affects phosphorus metabolism in the lens, resulting in: an increase of alpha-glycerophosphate (GP); an increase of a 6-ppm peak (its composition has yet to be identified); and a decrease in ATP level. These changes can be prevented by including sorbinil, an aldose reductase inhibitor, in the incubating medium.14 In addition to these changes, partial loss of phosphorylcholine (PC), and disappearance of glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were also observed in the lens of streptozotocin-induced diabetic rats (Fig 2). The physiologic significance of these changes is largely unknown; some tentative explanations are presented on the following pages. Change in Alpha-Glycerophosphate
Level
An increase of GP in the diabetic lens suggests activation of the NADH-requiring GP dehydrogenase. The GP levels reached a new steady state several hours after incubation of lenses in high glucose.‘4 NADH is apparently supplied by polyol dehydrogenase as a result of sorbitol pathway activation (Fig 1). Lactate dehydrogenase does not appear to use the surplus NADH since no significant increase in lactate production in high-glucose lenses was observed. Thus, GP dehydrogenase appears to function as an NAD-regenerating step. Without this regeneration, glycolytic activity would be severely limited because of a lack of NAD available for glyceraldehyde-3-phosphate dehydrogenase activity (Fig 1). A similar increase of GP levels in lenses incubated in the presence of cyanide, which caused an increase of anaerobic glycolysis, was also observed. In this case, although lactate production increased by 50%,15 NAD regeneration still required the participation of GP dehydrogenase. Since the GP level reaches a steady state, it is possible to estimate the ratio of free (ie, unbound) NADH/NAD based on the equilibrium constant of GP dehydrogenase.16 This ratio is 1.35 in the control lenses (5.5 mmol/L glucose) and Metabolism, Vol 35, No 4,
Suppl 1 (April), 1986: pp lo-14
EFFECT OF HIGH GLUCOSE AND OXIDATIVE
STRESS
11
PC
LENS
wNTP-------
I 0
include a 45” pulse width, 4-K data points, 5,000-Hz spectral width, 0.5 second interpulse delay, and proton decoupling. Samples were run at 34” C: 44,000 accumulations were averaged per spectrum. An exponential filter resulting in 1 Hz line-broadening was used. Resonance assignments: alpha GP, alpha-glycerophosphate: A, ribose-5-phosphate/AMP/IMP (resonance overlap): PC, phosphorylcholine; GPE, glycerophosphorylethanolamine: GPC. glycerophosphorylcholine; gamma, alpha, and beta NTP, gamma, alpha, and beta resonances of nucleotide triphosphate (mostly ATP); beta, alpha NDP, nucleotide diphosphate (mostly ADP). The change in ATP, GP, and 6-ppm component levels agrees with results from previous lens incubation studies.”
kinetic properties of other ATP-activated processes; for example, uptake of myo-inositol and amino acids may become less efficient. Since lens ATP is produced mainly from anaerobic glycolysis, which is regulated by the adenylate energy charge,‘* whether the new steady-state ATP level reflects a different charge level or a decrease in other, lesser ATP-generating activities is not known. An increase in the NADH/NAD
Metabolites
in the Human Lens
L.%ls
Age (vr)
NO.
Nondiabetic
71 ?6
14
0.15
* 0.11
0.06
+ 0.06
0.10
* 0.07
18.1 _t 5.6
0.67
i 0.32
Diabetic
68 _t 6
11
0.45
* 0.29
0.33
+ 0.23
0.29
? 0.16
47.2
0.67
2 0.35
-
-
I -20
Fig 2. P-31 NMR spectra of control and diabetic rat lenses. Diabetes mellitus was induced with injection of streptozotocin (85 mglkg body weight, prepared in O.Ol-mol/L citrate buffer, pli 4.5) through the tail vein. Blood glucose was 368 mg/dL and 117 mg/dL in the diabetic and control rats, respectively, at the time of sacrifice (7 days). Extracts were prepared with 10% perchloric acid (PCA): homogenization and acidification of lenses were conducted at low temperature with liquid nitrogen. Lyophilizates of PCA extracts from eight normal and eight diabetic crystalline lenses were reconstituted at a concentration of 0.4 gm wet weight/ml with an NMR solution of 20 mmol/L EDTA and 20% 40, pli 9.0. P-31 NMR spectroscopy was conducted at 109.3 M/H, using a Bruker HX270 spectrometer. NMR parameters
A decrease in the ATP level was observed in lenses incubated in high glucose’4 and in lenses from diabetic rats (Fig 2). Although it is possible that the decrease signifies an accelerated ATP consumption because of increased lens ionic pump activity, no increase in ADP or AMP levels was found-l4 On the other hand, the low ATP level remained constant despite a linear increase of sorbitol during highglucose incubation. This seems to indicate that a maximal pr,lduction and consumption of ATP has been reached. A Limited energy capacity to resist osmotic stress is therefore mplied. Low ATP and/or its availability can also alter the
GlWXe*
I -10 6 (PPM)
oj‘ A TP Level
Table 1. Sorbitol Pathway
LENS A.. -
2.:;4 in high-glucose (35.5 mmol/L) lenses. With the addition of sorbinil, the latter value is reduced to 0.76. In lenses incubated in the presence of cyanide, the ratio is 2.28. The fate of GP is unclear; however, since lens GP reaches a new steady state after incubation in a high-glucose medium, mechanisms facilitating utilization of GP must exist. There arc several possibilities: the GP cycle may be active in the lens; GP may be a secondary substrate reserve during energy deprivation; and GP may participate in phospholipid synthesis. A similar increase in GP levels is also found in human diabetic lenses (Table I). This is indirect evidence of increased flux through polyol dehydrogenase. Change
DIABETfC
QGP 1
Fig 1. Schematic representation of interacting metabolic pathways in the lens. Interactions are through cofactors and adenylates (for review on the effect of the adenylates. see ref. 12). I1 1Aldose reductase; (2) polyol dehydrogenase; (3) hexokinase: (4) ketohexokinase: (5) hexokinase; (61 glucose phosphate isomerese: (7) phosphofructokinase; (8) aldolase: (9) triose phosphate isomerase: (10) alpha-glycerophosphate dehydrogenase; (11) glyceraldehyde-l-phosphate dehydrogenase; (12) lactate dehydrogenase; (13) glucose-6-phosphate dehydrogenase; 114) glutathione reductase; and (15) glutathione peroxidase.
Swbitol*
Fructose*
GPt
+ 21.3
DHAPt
l/.unol/le”s. tnmol/lens. Lenses were obtained from the National Diabetes Research Interchange, Philadelphia, Pa. Assay of metabolites was performed according to Bergmeyer.”
Data show increased metabolite levels in the diabetic lens with the exception of dihydroxyacetone phosphate (DHAP). Increase I”
alpha-glycerophosphate (GPI was also demonstrated with P-31 NMR spectroscopy in a separate study (Cheng HM, Gonzilez RG. unpublished data). Calculated difference in [glucose + sorbitol + fructose] between diabetic and nondiabetic lenses is 0.76 @mol/lens, which is equivalent to a net increase of 5 mOsm in the diabetic lens (based on 60% water, 250 mg lens wet weight).
CHENG
ratio may inhibit respiration: oxygen uptake has been shown to decrease in diabetic peripheral nerves.” Whether this occurs in the lens has yet to be determined. Membrane Change A change in PC, GPC, and GPE levels in the diabetic rat lens suggests an alteration in membrane metabolism. The decrease in PC can be attributed to a decrease in ATP availability for the phosphorylation of choline. However, the loss of GPC and GPE cannot be explained at present. It is known that both permeability and transport are modified in the diabetic lens. Detailed mechanisms are presently unclear; for example, how does an increase in lens osmolarity per se alter membrane structure/function? Also, is there a change in membrane metabolism involving phospholipid/cholesterol synthesis, and is there a correlation between disturbance in membrane metabolism and structure/function? REDOX STATE
Perhaps the most intriguing change is the redox state in the lens. Activation of the sorbitol pathway involves rapid turnover of the cofactors. The effect of a change in the NADH/NAD ratio has been discussed above. The significance of competition for NADPH among various metabolic functions is detailed below.
With C-13 NMR spectroscopy, it is possible to perform “pulse-chase” studies to determine the fluxes of glucose, sorbitol, and fructose through metabolic pathways in the lens.19 In these studies, lenses are incubated in a medium containing [13C]-enriched glucose to allow a buildup of labeled glucose, sorbitol, and fructose in the lens. The lenses are then transferred to a medium containing unenriched glucose. The rate of disappearance of [13C] labels allows a determination of the flux rate of each metabolite. It is now known that one third of glucose is channeled through the sorbitol pathway in rabbit lenses incubated in 35.5 mmol/L glucose. The turnover rate of NADPH to NADP was estimated to be 3,000%/h and of NAD to NADH, 60%/h.19 These values may represent a low estimate because the Table 2. Sorbitol Pathway GlUCOSe
Fluxes*
Sorbitol
12 24
49.2
36
104.4
+ 1.4
15.7 * 1.2
+ 10.2
38.2
f 3.5
1.7 + 0.4
1.7 + 0.3
7.7 k 1.0
8.1 c 1.0
26.0
calculations were based on total (ie, free + bound) cofactors; only the free forms participate in redox reactions. In a more detailed study, the flux rates of glucose, sorbitol, and fructose in rat lenses were documented (Table 2). The data confirmed that a substantial portion of total glucose flux was through the sorbitol pathway at all three glucose concentrations tested. Furthermore, the rate-limiting step in the sorbitol pathway appears to be polyol dehydrogenase; this explains the accumulation of sorbitol of 6.2, 8.0, and 12.2 nmol/h/lens at glucose concentrations of 12, 24, and 36 mmol/L, respectively. It was not possible to determine the flux rates at 5.5 mmol/L glucose because C-13 NMR spectroscopy is not sensitive enough at this glucose level. Nevertheless, from what is known about the correlation between glucose concentration and the HMPS activity, one can infer that the flux rates at 5.5 mmol/L glucose are roughly one sixth of those at 35.5 mmol/L glucose. The glucose to sorbitol turnover rate agrees well with data based on HMPS activity, eg, at 32 mmol/L glucose, the rate of sorbitol production in a 25-mg rat lens is 33.6 nmol/h/lens,*’ which compares favorably with 38.2 nmol/h/lens (at 36 mmol/L glucose) determined with NMR spectroscopy (Table 2). The turnover rate of NADPH to NADP in the human lens can also be estimated. Table 3 shows a linear increase of the HMPS in human lens with increasing glucose concentration in the incubating medium. At 35.5 mmol/L glucose, the calculated rate of sorbitol production is 19.64 nmol/h/lens or 0.5 pmol/d, or l/14 the rate based on aldose reductase kinetics.* Using this rate and the NADP(H) content in the lens,8 the turnover rate of NADPH to NADP was determined to be 900%/h. Oxidative Resistance The prodigious use of NADPH may hamper the diabetic lens’s ability to maintain the sulfhydryl state through the NADPH-dependent glutathione reductase/peroxidase system.*‘~** The presence of hydrogen peroxide (HP) in the aqueous humor and the lens has recently been demonstrated.*’ Thus, the HMPS is constantly activated to provide NADPH.** Interference of HP detoxification by aldose reductase activation is substantiated by a study shown in Fig 3. The rate of HP removal by the lens is a linear function of HP concentration in the incubating medium; this rate is
Other
Fructose
7.9 f 0.6
14.8 k 2.4
+ 3.4
26.0
Table 3. Hexose Monophosphate
*Values
shown
NMR
Rat lenses
This
incubated
overnight
rate
plus
the
turnover
rate
medium
containing
the rates
of C-l
recorded. fructose
-
rate
of glucose
other,
was
rates
performed
in medium
and
the
-
fructose The
glucose
disappearance
respectively.
containing
-
sorbitol.
represent
according
accumulation
of sorbitol
unenriched
3 label
These
NADPH
PIMPS
Glucose
+ 3.4
No.
Productionf
Activity*
are nmol/h/lens.
spectroscopy
were
?,-glucose
Shunt (HMPSI Activity
in the Human Lens
LmmollL) C-13
GONZALEZ
Fructose -
Sorbitol-
Glucose * Glucose flux
(mmol/LI
AND
lenses at the
from
glucose
12. rate
24,
or 36
of sorbitol
turnover were three
glucose, flux,
to Gonzalez
mmol/L
transferred
concentrations sorbitol,
sorbitol
-
18
3.95
+ 0.85
6
9.82
t
60.0
6
15.52
recorded.
represents
then
5.5 35.5
et al.19
the to and
and fructose fructose,
and
lnmol glucose tnmol
NADPH
Human setts
lenses
Kinoshita
was
k 4.93
31.04
produced/h/lens. were
determined
et al.‘a
19.64
consumed/h/lens.
collected
Eye and Ear Infirmary
activity
7.90
3.85
in the operating
and used within using
“C,-glucose
rooms
2 hours
at the Massachu-
after
following
surgery. the
HMPS
methods
of
EFFECT OF HIGH GLUCOSE AND OXIDATIVE
13
STRESS
Table 4. HMPS Activity in Lenses Treated With 40 pmol/L Sorbinil Lens
Rat
10 -
Human
lnmol/h/lens.
Sorbinil
HMPS*
_
2.6 ? 0.4t
8
+
0.9 + 0.2t
8
.-
4.0 k 0.9
+
2.5 + O.?$
Lenses were incubated
NO
18 4
at 37.5 ‘C in 5.5 mmol/L
glucose.
. .
5.5 rn~ Glucose 35 5 mM Glucose
q
35 5mM
Glucose+Sorb~nd
I
0
02
04
06
08
10
15
.20
H202cont. I” medium ImMl Fig 3. The rate of hydrogen peroxide detoxification in the lens. Lenses were incubated in Dulbecco’s phosphate-buffered saline supplemented with 5.5 or 35.5 mmol/L glucose. Sorbinil. an aldose reductase inhibitor, was used at 40 pmol/L. Four rat lenses were incubated in 2 mL of medium containing various concentrations of hydrogen peroxide (HP) from 0 to 0.2 mmol/L. Cat&se activity was monitored with a Clark oxygen probe; the activity was detected only at HP concentrations of more than 0.2 mmol/L. The rate of HP disappearance from the medium was determined by assaying initial and final [HP] in a 20-minute incubating period. HP was measured according to Giblin et aI.= Each data point represents the average of four groups of lenses (four lenses in each
group).
decreased in lenses incubated in 35.5 mmol/L glucose and is reversed with the addition of sorbinil (Fig 3). It should be noted that there is no evidence of thiol regeneration through dc novo synthesis of GSH; furthermore, although direct interaction between oxidants and NADPH may occur, there is no activation of the hexose monophosphate shunt through such an interaction. These have been previously shown in a study using I-chloro-2,4-dinitrobenzene, which coupled with GSH through GSH S-transferase.24 It is also conceivable that the consumption of NADPH for sorbitol production and for thiol maintenance may affect
tttest:
P<
tttest:
P < 0.01.
0.001.
other NADPH-requiring mechanisms, such as fatty acid and cholesterol synthesis. Sorbitol pathway flux is decreased in lenses incubated in high glucose (35.5 mmol/L) with 0.1 mmol/L HP to a rate of 5.7 + 0.8 nmol/h/lens, which is comparable to that of lenses incubated in an 8-mmol/L glucose medium. This shows that the sorbitol pathway still channels away a significant amount of NADPH even when the lens is under severe oxidative stress and needs NADPH for thiol maintenance. It should be noted that the sorbitol pathway is operative at low glucose levels. Sorbinil, at 40 Fmol/L, inhibits sorbitol production (Hutson N, personal communication) with a concomitant decrease of HMPS activity (Table 4) in lenses incubated in 5.5 mmol/L glucose. A possible role for aldose reductase inhibitors in enhancing the lens’s oxidative resistance, by decreasing competition for NADPH by aldose reductase. is suggested. CONCLUSIONS
Clearly, diabetic cataractogenesis is a multifactorial process. In addition to osmotic stress, loss of energy, disturbance to lens membrane integrity and its function, and decreased oxidative resistance are possible contributing factors. Even if the accumulation of sorbitol is not osmotically significant, there is still the extremely rapid flux of glucose through the sorbitol pathway, which will cause a shift of cofactor ratios as well as a change in energy generation/consumption. Thus, glucose flux through the sorbitol pathway may be the fundamental underlying mechanism of diabetic complications. However, it is also evident from our data that aldose reductase inhibitors can prevent metabolic changes in the diabetic lens. ACKNOWLEDGMENT
We wish to thank Patrick Barnett, MD, lngrid von Saltza, Buckley, and Stefano Miglior, MD, for their assistance.
Lisa
REFERENCES 1. Kinoshita JH, Kador P, Datiles M: Aldose reductase in di:rbetic cataracts. J Am Med Assoc 246:257-26 1, 198 1 2. Kinoshita JH: Mechanisms initiating cataract formation. Invest Ophthalmol 13:713-724, 1974 3. Chylack LT Jr, Kinoshita JH: A biochemical evaluation of cataract induced in high glucose medium. Invest Ophthalmol8:401~ 412. 1969 4. Pirie A, van Heyningen R: The effect of diabetes on the content
of sorbitol, glucose, fructose and inositol in the human lens. Exp Eye Res 3:124-131,1964 5. Varma SD, Kinoshita JH: Sorbitol pathway in diabetic and galactosemic rat lens. Biochim Biophys Acta 338:632-640, 1974 6. Varma SD, Schocket SS, Richards RD: Implications of aldose reductase in cataracts in human diabetes. Invest Ophthalmol Vis Sci 18:237-241. 1979 7. Chylack LT Jr. Henriquez HF 111. Cheng HM. et al: Efficacy
14
of alrestatin, an aldose reductase inhibitor in human diabetic and non-diabetic lenses. Ophthalmol86:1579-1585, 1979 8. Jedziniak JA, Chylack LT Jr, et al: The sorbitol pathway in the human lens: Aldose reductase and polyol dehydrogenase. Invest Ophthalmol Vis Sci 20:3 14-326, 1984 9. Akagi Y, Yajima Y, Kador PF, et al: Localization of aldose reductase in the human eye. Diabetes 33:562-566, 1984 10. Jacob TJC, Duncan Cl: Glucose-induced membrane permeability changes in the lens. Exp Eye Res 34445453, 1982 1 I. Cheng HM, Gonzalez RG, Barnett PA, et al: Sorbitol/ fructose metabolism in the lens. Exp Eye Res 40:223-229, 1985 12. Cheng HM, Chylack LT Jr: Lens metabolism, in Maisel H (ed): The Ocular Lens. New York, Marcel-Dekker, 1985, p 223 13. Kinoshita JH, Futterman S, Satoh K, et al: Factors affecting the formation of sugar alcohols in ocular lens. Biochim Biophys Acta 74:340-350.1963 14. Gonzalez RG, Barnett PA, Cheng HM, et al: Altered phosphate metabolism in the lens exposed to high glucose and its prevention by an aldose reductase inhibitor. Exp Eye Res 39:553562, 1984 15. Gillis MK, Chylack LT Jr, Cheng HM: Age and control of glycolysis in the rat lens. Invest Ophthalmol Vis Sci 20:457-466, 1981 16. von Saltza I, Gonzalez RG, Barnett PA, et al: Glycolytic flux
CHENG AND GONZALEZ
and the redox state of free dinucleotides publication)
in the lens. (submitted
17. Bergmeyer HU (ed): Methods of Enzymatic do, Fla, Academic, 1974
Analysis.
for
Orlan-
18. Greene DA, Winegrad Al: Effects of acute experimental diabetes on composite energy metabolism in peripheral nerve axons and Schwann cells. Diabetes 30:967-974, 1981 19. Gonzalez RG, Barnett PA, Aguayo JB, et al: Direct measurement of polyol pathway activity in the ocular lens. Diabetes 33:196199,1984 20. Cheng HM, Fagerholm P, Chylack LT Jr: Response of the lens to oxidative-osmotic stress. Exp Eye Res 37:1 l-21, 1983 21. Giblin FJ, Nies DE, Reddy VN: Stimulation of the hexose monophosphate shunt in rabbit lens in response to the oxidation of glutathione. Exp Eye Res 33:289-298, 1981 22. Giblin FJ, McCready JP, Reddy VN: The role of glutathione metabolism in the detoxification of H,O, in rabbit lens. Invest Ophthalmol Vis Sci 22:330-335, 1982 23. Spector A, Garner WH: cataracts. Exp Eye Res 33:673-68
Hydrogen 1, 1981
24. Cheng HM, von Saltza I, Gonzalez glutathione deprivation on lens metabolism. 365, 1984
peroxide
and
human
RG, et al: Effect of Exp Eye Res 39:355-
Cheng and R. Gilbert0
Gonzilez
Diabetic cataractogenesis, a multifactorial process, was examined with nuclear magnetic resonance (NMR). P-31 NMR spectroscopic studies showed substantial alteration of both energy and membrane metabolism in the diabetic lens. Findings from a C-13 NMR spectroscopic determination of the sorbitol pathway flux in lenses incubated in 35.5 mmol/L glucose revealed that (1) one-third of total glucose consumed was channeled through this pathway, and (2) the turnover rate of NADPH to NADP was 3,00O%/hr. Furthermore, a competition for NADPH between aldose reductase and glutathione reductase was demonstrated. It is important to note that all metabolic changes in hyperglycemic/diabetic lenses can be prevented by aldose reductase inhibitors, o 1986 by Grune & Stratton, inc.
eg, sorbinil.
T
HE INVOLVEMENT of the sorbitol pathway in diabetic cataractogenesis in animal models has long been established.‘-’ However, its role in diabetic human cataract formation is not as clear-cut. Sorbitol contents in postmortem diabetic human lenses, as well as in lenses incubated in high glucose, are much lower than those in experimental diabetic cataract models.“’ Based on aldose reductase kinetics, it was estimated that human lenses can generate 7 hmol sorbitol/lens/d at a glucose concentration of 35.5 mmol/L.’ This is, however, the maximal rate; in vivo production may be considerably less. Nonetheless. the osmotic stress theory may still apply if one argues that sorbitol accumulates in the epithelium and superficial cortex, where most aldose reductase activity resides. 8*9This is conceivable but difficult to verify. The osmotic stress issue is further complicated by extralenticular factors: (1) a concurrent increase of osmolarity in the aqueous and vitreous humors may offset the increase in lens tonicity; on the other hand, one can also argue that (2) the increase in aqueous glucose per se may be osmotically stressful to the lens,” particularly if the lens is incapable of compensating rapid fluctuation of glucose levels in the aqueous humor.” In tissues such as the peripheral nerve, kidney, and human lens that do not produce and accumulate large quantities of sorbitol, factors other than or in addition to the osmotic stress may also play a pathogenic role. In the animal lens model, these factors may have been masked by the acute osmotic imbalance caused by accumulation of lens sorbitol. We will examine several of these factors. PHOSPHORUS
METABOLISM
Activation of the sorbitol pathway is not an isolated event, ie, other interacting metabolic activities are also involved From the Howe Laboratory of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, Boston. Mass, and the Francis Biiter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Mass. Presented at the Pfizer Sorbinil Symposium-The Eflects of Sorbinil on the Pathophysiology of Diabetic Complications, Dorado, Puerto Rico, May 30-June 2, 1985. Supported by Grant EY04424 from the National Eye Institute. Address reprint requests to Hong-Ming Cheng, PhD, Howe Laboratory of Ophthalmology, 243 Charles St, Boston, MA 02114. o 1986 by Grune & Stratton, Inc. 0026-a495/86/3504-1003$03.00/O 10
(Fig 1). The interactions are mediated through cofactors and adenylates.” Stimulation of the hexose monophosphate shunt (HMPS) in conjunction with sorbitol pathway activation is well known.13 Other activities are less obvious but have become apparent after lens metabolism is evaluated with P-3 1 nuclear magnetic resonance (NMR) spectroscopy, which provides information on all phosphorus metabolites above 0.2 mmol/L in concentration (with present-day technology). P-31 NMR studies showed that activation of the sorbitol pathway affects phosphorus metabolism in the lens, resulting in: an increase of alpha-glycerophosphate (GP); an increase of a 6-ppm peak (its composition has yet to be identified); and a decrease in ATP level. These changes can be prevented by including sorbinil, an aldose reductase inhibitor, in the incubating medium.14 In addition to these changes, partial loss of phosphorylcholine (PC), and disappearance of glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were also observed in the lens of streptozotocin-induced diabetic rats (Fig 2). The physiologic significance of these changes is largely unknown; some tentative explanations are presented on the following pages. Change in Alpha-Glycerophosphate
Level
An increase of GP in the diabetic lens suggests activation of the NADH-requiring GP dehydrogenase. The GP levels reached a new steady state several hours after incubation of lenses in high glucose.‘4 NADH is apparently supplied by polyol dehydrogenase as a result of sorbitol pathway activation (Fig 1). Lactate dehydrogenase does not appear to use the surplus NADH since no significant increase in lactate production in high-glucose lenses was observed. Thus, GP dehydrogenase appears to function as an NAD-regenerating step. Without this regeneration, glycolytic activity would be severely limited because of a lack of NAD available for glyceraldehyde-3-phosphate dehydrogenase activity (Fig 1). A similar increase of GP levels in lenses incubated in the presence of cyanide, which caused an increase of anaerobic glycolysis, was also observed. In this case, although lactate production increased by 50%,15 NAD regeneration still required the participation of GP dehydrogenase. Since the GP level reaches a steady state, it is possible to estimate the ratio of free (ie, unbound) NADH/NAD based on the equilibrium constant of GP dehydrogenase.16 This ratio is 1.35 in the control lenses (5.5 mmol/L glucose) and Metabolism, Vol 35, No 4,
Suppl 1 (April), 1986: pp lo-14
EFFECT OF HIGH GLUCOSE AND OXIDATIVE
STRESS
11
PC
LENS
wNTP-------
I 0
include a 45” pulse width, 4-K data points, 5,000-Hz spectral width, 0.5 second interpulse delay, and proton decoupling. Samples were run at 34” C: 44,000 accumulations were averaged per spectrum. An exponential filter resulting in 1 Hz line-broadening was used. Resonance assignments: alpha GP, alpha-glycerophosphate: A, ribose-5-phosphate/AMP/IMP (resonance overlap): PC, phosphorylcholine; GPE, glycerophosphorylethanolamine: GPC. glycerophosphorylcholine; gamma, alpha, and beta NTP, gamma, alpha, and beta resonances of nucleotide triphosphate (mostly ATP); beta, alpha NDP, nucleotide diphosphate (mostly ADP). The change in ATP, GP, and 6-ppm component levels agrees with results from previous lens incubation studies.”
kinetic properties of other ATP-activated processes; for example, uptake of myo-inositol and amino acids may become less efficient. Since lens ATP is produced mainly from anaerobic glycolysis, which is regulated by the adenylate energy charge,‘* whether the new steady-state ATP level reflects a different charge level or a decrease in other, lesser ATP-generating activities is not known. An increase in the NADH/NAD
Metabolites
in the Human Lens
L.%ls
Age (vr)
NO.
Nondiabetic
71 ?6
14
0.15
* 0.11
0.06
+ 0.06
0.10
* 0.07
18.1 _t 5.6
0.67
i 0.32
Diabetic
68 _t 6
11
0.45
* 0.29
0.33
+ 0.23
0.29
? 0.16
47.2
0.67
2 0.35
-
-
I -20
Fig 2. P-31 NMR spectra of control and diabetic rat lenses. Diabetes mellitus was induced with injection of streptozotocin (85 mglkg body weight, prepared in O.Ol-mol/L citrate buffer, pli 4.5) through the tail vein. Blood glucose was 368 mg/dL and 117 mg/dL in the diabetic and control rats, respectively, at the time of sacrifice (7 days). Extracts were prepared with 10% perchloric acid (PCA): homogenization and acidification of lenses were conducted at low temperature with liquid nitrogen. Lyophilizates of PCA extracts from eight normal and eight diabetic crystalline lenses were reconstituted at a concentration of 0.4 gm wet weight/ml with an NMR solution of 20 mmol/L EDTA and 20% 40, pli 9.0. P-31 NMR spectroscopy was conducted at 109.3 M/H, using a Bruker HX270 spectrometer. NMR parameters
A decrease in the ATP level was observed in lenses incubated in high glucose’4 and in lenses from diabetic rats (Fig 2). Although it is possible that the decrease signifies an accelerated ATP consumption because of increased lens ionic pump activity, no increase in ADP or AMP levels was found-l4 On the other hand, the low ATP level remained constant despite a linear increase of sorbitol during highglucose incubation. This seems to indicate that a maximal pr,lduction and consumption of ATP has been reached. A Limited energy capacity to resist osmotic stress is therefore mplied. Low ATP and/or its availability can also alter the
GlWXe*
I -10 6 (PPM)
oj‘ A TP Level
Table 1. Sorbitol Pathway
LENS A.. -
2.:;4 in high-glucose (35.5 mmol/L) lenses. With the addition of sorbinil, the latter value is reduced to 0.76. In lenses incubated in the presence of cyanide, the ratio is 2.28. The fate of GP is unclear; however, since lens GP reaches a new steady state after incubation in a high-glucose medium, mechanisms facilitating utilization of GP must exist. There arc several possibilities: the GP cycle may be active in the lens; GP may be a secondary substrate reserve during energy deprivation; and GP may participate in phospholipid synthesis. A similar increase in GP levels is also found in human diabetic lenses (Table I). This is indirect evidence of increased flux through polyol dehydrogenase. Change
DIABETfC
QGP 1
Fig 1. Schematic representation of interacting metabolic pathways in the lens. Interactions are through cofactors and adenylates (for review on the effect of the adenylates. see ref. 12). I1 1Aldose reductase; (2) polyol dehydrogenase; (3) hexokinase: (4) ketohexokinase: (5) hexokinase; (61 glucose phosphate isomerese: (7) phosphofructokinase; (8) aldolase: (9) triose phosphate isomerase: (10) alpha-glycerophosphate dehydrogenase; (11) glyceraldehyde-l-phosphate dehydrogenase; (12) lactate dehydrogenase; (13) glucose-6-phosphate dehydrogenase; 114) glutathione reductase; and (15) glutathione peroxidase.
Swbitol*
Fructose*
GPt
+ 21.3
DHAPt
l/.unol/le”s. tnmol/lens. Lenses were obtained from the National Diabetes Research Interchange, Philadelphia, Pa. Assay of metabolites was performed according to Bergmeyer.”
Data show increased metabolite levels in the diabetic lens with the exception of dihydroxyacetone phosphate (DHAP). Increase I”
alpha-glycerophosphate (GPI was also demonstrated with P-31 NMR spectroscopy in a separate study (Cheng HM, Gonzilez RG. unpublished data). Calculated difference in [glucose + sorbitol + fructose] between diabetic and nondiabetic lenses is 0.76 @mol/lens, which is equivalent to a net increase of 5 mOsm in the diabetic lens (based on 60% water, 250 mg lens wet weight).
CHENG
ratio may inhibit respiration: oxygen uptake has been shown to decrease in diabetic peripheral nerves.” Whether this occurs in the lens has yet to be determined. Membrane Change A change in PC, GPC, and GPE levels in the diabetic rat lens suggests an alteration in membrane metabolism. The decrease in PC can be attributed to a decrease in ATP availability for the phosphorylation of choline. However, the loss of GPC and GPE cannot be explained at present. It is known that both permeability and transport are modified in the diabetic lens. Detailed mechanisms are presently unclear; for example, how does an increase in lens osmolarity per se alter membrane structure/function? Also, is there a change in membrane metabolism involving phospholipid/cholesterol synthesis, and is there a correlation between disturbance in membrane metabolism and structure/function? REDOX STATE
Perhaps the most intriguing change is the redox state in the lens. Activation of the sorbitol pathway involves rapid turnover of the cofactors. The effect of a change in the NADH/NAD ratio has been discussed above. The significance of competition for NADPH among various metabolic functions is detailed below.
With C-13 NMR spectroscopy, it is possible to perform “pulse-chase” studies to determine the fluxes of glucose, sorbitol, and fructose through metabolic pathways in the lens.19 In these studies, lenses are incubated in a medium containing [13C]-enriched glucose to allow a buildup of labeled glucose, sorbitol, and fructose in the lens. The lenses are then transferred to a medium containing unenriched glucose. The rate of disappearance of [13C] labels allows a determination of the flux rate of each metabolite. It is now known that one third of glucose is channeled through the sorbitol pathway in rabbit lenses incubated in 35.5 mmol/L glucose. The turnover rate of NADPH to NADP was estimated to be 3,000%/h and of NAD to NADH, 60%/h.19 These values may represent a low estimate because the Table 2. Sorbitol Pathway GlUCOSe
Fluxes*
Sorbitol
12 24
49.2
36
104.4
+ 1.4
15.7 * 1.2
+ 10.2
38.2
f 3.5
1.7 + 0.4
1.7 + 0.3
7.7 k 1.0
8.1 c 1.0
26.0
calculations were based on total (ie, free + bound) cofactors; only the free forms participate in redox reactions. In a more detailed study, the flux rates of glucose, sorbitol, and fructose in rat lenses were documented (Table 2). The data confirmed that a substantial portion of total glucose flux was through the sorbitol pathway at all three glucose concentrations tested. Furthermore, the rate-limiting step in the sorbitol pathway appears to be polyol dehydrogenase; this explains the accumulation of sorbitol of 6.2, 8.0, and 12.2 nmol/h/lens at glucose concentrations of 12, 24, and 36 mmol/L, respectively. It was not possible to determine the flux rates at 5.5 mmol/L glucose because C-13 NMR spectroscopy is not sensitive enough at this glucose level. Nevertheless, from what is known about the correlation between glucose concentration and the HMPS activity, one can infer that the flux rates at 5.5 mmol/L glucose are roughly one sixth of those at 35.5 mmol/L glucose. The glucose to sorbitol turnover rate agrees well with data based on HMPS activity, eg, at 32 mmol/L glucose, the rate of sorbitol production in a 25-mg rat lens is 33.6 nmol/h/lens,*’ which compares favorably with 38.2 nmol/h/lens (at 36 mmol/L glucose) determined with NMR spectroscopy (Table 2). The turnover rate of NADPH to NADP in the human lens can also be estimated. Table 3 shows a linear increase of the HMPS in human lens with increasing glucose concentration in the incubating medium. At 35.5 mmol/L glucose, the calculated rate of sorbitol production is 19.64 nmol/h/lens or 0.5 pmol/d, or l/14 the rate based on aldose reductase kinetics.* Using this rate and the NADP(H) content in the lens,8 the turnover rate of NADPH to NADP was determined to be 900%/h. Oxidative Resistance The prodigious use of NADPH may hamper the diabetic lens’s ability to maintain the sulfhydryl state through the NADPH-dependent glutathione reductase/peroxidase system.*‘~** The presence of hydrogen peroxide (HP) in the aqueous humor and the lens has recently been demonstrated.*’ Thus, the HMPS is constantly activated to provide NADPH.** Interference of HP detoxification by aldose reductase activation is substantiated by a study shown in Fig 3. The rate of HP removal by the lens is a linear function of HP concentration in the incubating medium; this rate is
Other
Fructose
7.9 f 0.6
14.8 k 2.4
+ 3.4
26.0
Table 3. Hexose Monophosphate
*Values
shown
NMR
Rat lenses
This
incubated
overnight
rate
plus
the
turnover
rate
medium
containing
the rates
of C-l
recorded. fructose
-
rate
of glucose
other,
was
rates
performed
in medium
and
the
-
fructose The
glucose
disappearance
respectively.
containing
-
sorbitol.
represent
according
accumulation
of sorbitol
unenriched
3 label
These
NADPH
PIMPS
Glucose
+ 3.4
No.
Productionf
Activity*
are nmol/h/lens.
spectroscopy
were
?,-glucose
Shunt (HMPSI Activity
in the Human Lens
LmmollL) C-13
GONZALEZ
Fructose -
Sorbitol-
Glucose * Glucose flux
(mmol/LI
AND
lenses at the
from
glucose
12. rate
24,
or 36
of sorbitol
turnover were three
glucose, flux,
to Gonzalez
mmol/L
transferred
concentrations sorbitol,
sorbitol
-
18
3.95
+ 0.85
6
9.82
t
60.0
6
15.52
recorded.
represents
then
5.5 35.5
et al.19
the to and
and fructose fructose,
and
lnmol glucose tnmol
NADPH
Human setts
lenses
Kinoshita
was
k 4.93
31.04
produced/h/lens. were
determined
et al.‘a
19.64
consumed/h/lens.
collected
Eye and Ear Infirmary
activity
7.90
3.85
in the operating
and used within using
“C,-glucose
rooms
2 hours
at the Massachu-
after
following
surgery. the
HMPS
methods
of
EFFECT OF HIGH GLUCOSE AND OXIDATIVE
13
STRESS
Table 4. HMPS Activity in Lenses Treated With 40 pmol/L Sorbinil Lens
Rat
10 -
Human
lnmol/h/lens.
Sorbinil
HMPS*
_
2.6 ? 0.4t
8
+
0.9 + 0.2t
8
.-
4.0 k 0.9
+
2.5 + O.?$
Lenses were incubated
NO
18 4
at 37.5 ‘C in 5.5 mmol/L
glucose.
. .
5.5 rn~ Glucose 35 5 mM Glucose
q
35 5mM
Glucose+Sorb~nd
I
0
02
04
06
08
10
15
.20
H202cont. I” medium ImMl Fig 3. The rate of hydrogen peroxide detoxification in the lens. Lenses were incubated in Dulbecco’s phosphate-buffered saline supplemented with 5.5 or 35.5 mmol/L glucose. Sorbinil. an aldose reductase inhibitor, was used at 40 pmol/L. Four rat lenses were incubated in 2 mL of medium containing various concentrations of hydrogen peroxide (HP) from 0 to 0.2 mmol/L. Cat&se activity was monitored with a Clark oxygen probe; the activity was detected only at HP concentrations of more than 0.2 mmol/L. The rate of HP disappearance from the medium was determined by assaying initial and final [HP] in a 20-minute incubating period. HP was measured according to Giblin et aI.= Each data point represents the average of four groups of lenses (four lenses in each
group).
decreased in lenses incubated in 35.5 mmol/L glucose and is reversed with the addition of sorbinil (Fig 3). It should be noted that there is no evidence of thiol regeneration through dc novo synthesis of GSH; furthermore, although direct interaction between oxidants and NADPH may occur, there is no activation of the hexose monophosphate shunt through such an interaction. These have been previously shown in a study using I-chloro-2,4-dinitrobenzene, which coupled with GSH through GSH S-transferase.24 It is also conceivable that the consumption of NADPH for sorbitol production and for thiol maintenance may affect
tttest:
P<
tttest:
P < 0.01.
0.001.
other NADPH-requiring mechanisms, such as fatty acid and cholesterol synthesis. Sorbitol pathway flux is decreased in lenses incubated in high glucose (35.5 mmol/L) with 0.1 mmol/L HP to a rate of 5.7 + 0.8 nmol/h/lens, which is comparable to that of lenses incubated in an 8-mmol/L glucose medium. This shows that the sorbitol pathway still channels away a significant amount of NADPH even when the lens is under severe oxidative stress and needs NADPH for thiol maintenance. It should be noted that the sorbitol pathway is operative at low glucose levels. Sorbinil, at 40 Fmol/L, inhibits sorbitol production (Hutson N, personal communication) with a concomitant decrease of HMPS activity (Table 4) in lenses incubated in 5.5 mmol/L glucose. A possible role for aldose reductase inhibitors in enhancing the lens’s oxidative resistance, by decreasing competition for NADPH by aldose reductase. is suggested. CONCLUSIONS
Clearly, diabetic cataractogenesis is a multifactorial process. In addition to osmotic stress, loss of energy, disturbance to lens membrane integrity and its function, and decreased oxidative resistance are possible contributing factors. Even if the accumulation of sorbitol is not osmotically significant, there is still the extremely rapid flux of glucose through the sorbitol pathway, which will cause a shift of cofactor ratios as well as a change in energy generation/consumption. Thus, glucose flux through the sorbitol pathway may be the fundamental underlying mechanism of diabetic complications. However, it is also evident from our data that aldose reductase inhibitors can prevent metabolic changes in the diabetic lens. ACKNOWLEDGMENT
We wish to thank Patrick Barnett, MD, lngrid von Saltza, Buckley, and Stefano Miglior, MD, for their assistance.
Lisa
REFERENCES 1. Kinoshita JH, Kador P, Datiles M: Aldose reductase in di:rbetic cataracts. J Am Med Assoc 246:257-26 1, 198 1 2. Kinoshita JH: Mechanisms initiating cataract formation. Invest Ophthalmol 13:713-724, 1974 3. Chylack LT Jr, Kinoshita JH: A biochemical evaluation of cataract induced in high glucose medium. Invest Ophthalmol8:401~ 412. 1969 4. Pirie A, van Heyningen R: The effect of diabetes on the content
of sorbitol, glucose, fructose and inositol in the human lens. Exp Eye Res 3:124-131,1964 5. Varma SD, Kinoshita JH: Sorbitol pathway in diabetic and galactosemic rat lens. Biochim Biophys Acta 338:632-640, 1974 6. Varma SD, Schocket SS, Richards RD: Implications of aldose reductase in cataracts in human diabetes. Invest Ophthalmol Vis Sci 18:237-241. 1979 7. Chylack LT Jr. Henriquez HF 111. Cheng HM. et al: Efficacy
14
of alrestatin, an aldose reductase inhibitor in human diabetic and non-diabetic lenses. Ophthalmol86:1579-1585, 1979 8. Jedziniak JA, Chylack LT Jr, et al: The sorbitol pathway in the human lens: Aldose reductase and polyol dehydrogenase. Invest Ophthalmol Vis Sci 20:3 14-326, 1984 9. Akagi Y, Yajima Y, Kador PF, et al: Localization of aldose reductase in the human eye. Diabetes 33:562-566, 1984 10. Jacob TJC, Duncan Cl: Glucose-induced membrane permeability changes in the lens. Exp Eye Res 34445453, 1982 1 I. Cheng HM, Gonzalez RG, Barnett PA, et al: Sorbitol/ fructose metabolism in the lens. Exp Eye Res 40:223-229, 1985 12. Cheng HM, Chylack LT Jr: Lens metabolism, in Maisel H (ed): The Ocular Lens. New York, Marcel-Dekker, 1985, p 223 13. Kinoshita JH, Futterman S, Satoh K, et al: Factors affecting the formation of sugar alcohols in ocular lens. Biochim Biophys Acta 74:340-350.1963 14. Gonzalez RG, Barnett PA, Cheng HM, et al: Altered phosphate metabolism in the lens exposed to high glucose and its prevention by an aldose reductase inhibitor. Exp Eye Res 39:553562, 1984 15. Gillis MK, Chylack LT Jr, Cheng HM: Age and control of glycolysis in the rat lens. Invest Ophthalmol Vis Sci 20:457-466, 1981 16. von Saltza I, Gonzalez RG, Barnett PA, et al: Glycolytic flux
CHENG AND GONZALEZ
and the redox state of free dinucleotides publication)
in the lens. (submitted
17. Bergmeyer HU (ed): Methods of Enzymatic do, Fla, Academic, 1974
Analysis.
for
Orlan-
18. Greene DA, Winegrad Al: Effects of acute experimental diabetes on composite energy metabolism in peripheral nerve axons and Schwann cells. Diabetes 30:967-974, 1981 19. Gonzalez RG, Barnett PA, Aguayo JB, et al: Direct measurement of polyol pathway activity in the ocular lens. Diabetes 33:196199,1984 20. Cheng HM, Fagerholm P, Chylack LT Jr: Response of the lens to oxidative-osmotic stress. Exp Eye Res 37:1 l-21, 1983 21. Giblin FJ, Nies DE, Reddy VN: Stimulation of the hexose monophosphate shunt in rabbit lens in response to the oxidation of glutathione. Exp Eye Res 33:289-298, 1981 22. Giblin FJ, McCready JP, Reddy VN: The role of glutathione metabolism in the detoxification of H,O, in rabbit lens. Invest Ophthalmol Vis Sci 22:330-335, 1982 23. Spector A, Garner WH: cataracts. Exp Eye Res 33:673-68
Hydrogen 1, 1981
24. Cheng HM, von Saltza I, Gonzalez glutathione deprivation on lens metabolism. 365, 1984
peroxide
and
human
RG, et al: Effect of Exp Eye Res 39:355-