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The effects of long-term treatment of streptozotocin-diabetic rats with an aldose reductase inhibitor
E.xp.Eye Res.(1983)37,507-515
The Effects tozotocin-diabetic
R.POULSOM*,
Depurtment
of Long-term ats with ~~b~bit~r R.
Treatment an Aldose
of Reductase
$.BooT-HA~DFORD~ANDH.HEATH
of Biochemical Pathology! University College School of Medicine, University Street, London WClE 6JJ (Received 7 March
1983 and accepted 12 April
1983)
To test the possible involvement of the sorbitol pathway in the pathogenesis of retinopathy in Long-term experimentally-diabetic rats, streptozotocin-diabetic and normal rats were dosed orally (50 mg/kg body weight daily) for ub to 373 days with an aldose reductase inhibitor (ICI 105552) or a placebo. Long-term treatment with ICI 105552 (1,(3,4-dichlorobenzyl)-3-methyl-l,2-dihydro-2-oxoquinol-4.ylacetic acid; sodium salt) markedly reduced the normal accumulations of sorbitol and fructose in the sciatic nerves (86 and 69 y0 reductions) and seminal vesicles with coagulating glands (75 and 49 % reductions). Thus. by these criteria, the inhibitor was as effective after several months of dosing as after three weeks. There was no evidence that treatment with this aldose reductase inhibitor had any protective effect against the development of pathological changes in the retina and kidney of these rats. Key words: aldose reductase inhibitor: ICI 105552 ; sorbitol ; fructose; inositol ; streptozotocindiabetes; diabetic retinopathy.
I. l[ntroduction Aldose reduetase (E.C. 1.1 1.21) is the first enzyme of the sorbitol pathway, which converts glucose into sorbitol and then into fructose. Abnormally increased activity of this pathway is considered to be a contributing factor in the pathogenesis of cataract (Kinoshita, Kador and Datiles, 1981) and polyneuropathy (Fagius and Jameson, 1984 ; Judzewitsch et al., 1981) in diabetes. An early experiment (Leuenberger and Babel, 1978) t’o test the hypothesis (Beaumont, Hollows, Schofield, Williams and Steinbeck, 1971; Heath and Hamlett, 1976) that the sorbitol pathway might be involved in the development of microvascular complications in diabetes was inconclusive since quercetin, the aldose reductase inhibitor (ARI) with which they treated their diabetic rats: had no demonstrable effects in vivo even though it was administered as a 5 o!0 (w/w) admixture to the diet. Quercetin has a moderate activity in vitro (Varma, Mikuni and Kinoshita, 1975) and rats (Beyer-Mears and measurable effects when given orally to galactosaemic Farnsworth, 1979). The present study was designed to test the hypothesis by treating streptozotocindiabetic (SZ-diabetic) rats with a new orally-active aldose reductase inhibitor, ICI 105552, for 12 months. The SZ-diabetic rat develops some of the retinal lesions of human diabetic retinopathy in six to 12 months. These changes include tortuosity and irregularity of capillaries, preferential loss of intra-mural pericytes (mural cells), acellular capillaries, increased PAS-positive deposits, thickening of capillary basement * Please address correspondence to Dr R. Poulsom, Departments of Visual Science Institute of Ophthalmology, Judd Street, London WClH QQS G.K. 7 Present address: Department of Medical Biochemistry, University of Manchester Stopford Building, Oxford Road, Manchester Ml3 QPT C.K.
001&4835/83/010507+10
SOS.OO/O
0
1983 Academic
and Pathology, Medical
Press Inc. (London)
School,
Limited
R. POULSOM
508
ET
AL.
membranes, strand formation and, infrequently, fusiform or saccular microaneurysms (Cameron et al., 1971; Babel and Leuenberger, 1974; Kojima, Harada, Hoshino and Kojima, 1975; Papachristodoulou, Heath and Kang, 1976; Leuenberger and Babel, 1978; Boot-Handford and Heath, 1980). It was anticipated that during the course of the experiment the inhibitor would prevent or retard cataractogenesis; this was so and these results have been reported and discussed previously (Poulsom, Boot-Handford and Heath, 1982). In order to demonstrate that the aldose reductase inhibitor retained its effectiveness after many months of dosing the concentrations of glucose, sorbitol and fructose were measured in tissues known to possess the sorbitol pathway and these results were compared with those from a short-term study (Poulsom and Heath, 1983). 2. Materials Animals
and
Methods
and treatments
These details have been reported previously (Poulsom et al., 1982). Briefly, male (299 g) Wistar rats, housed in wire-bottomed cages with a standard diet (MRC 4lB) and water available ad libitum were assigned randomly to one of the four treatments; diabetic dosed with ICI 105552 (designated as treatment DA), diabetic dosed with placebo (DB), control dosed with ICI 105552 (CA), control dosed with placebo (CB). Diabetes was induced with streptozotocin (i.v.) and was confirmed if glucosuria and a fed-state blood glucose level > 16-6 mM were present one week later; these criteria were used periodically to confirm the persistence of diabetes. When necessary, further doses of the diabetogen were given (Poulsom, 1982). One week after the induction of diabetes once-daily dosage with the enzyme inhibitor or placebo as appropriate was commenced. Rats, originally 12 per treatment, were dosed at 50 mg/kg body weight by gastric intubation with a 25 g/l solution in deionized distilled water for up to 373 days. Preparation
of tissues
Two rats per day were anaesthetized deeply (Sagatal, May & Baker; 6&72 mg/kg body weight) before noon and at least 45 min after their last oral dose of ICI 105552 or placebo. Enzymatic or gas-liquid chromatographic methods (Poulsom and Heath, 1983) were used to assay glucose in tail vein blood and sugars and polyols in samples of the left kidney, the sciatic nerves and the seminal vesicles with attached coagnlating glands (SVCG). The eyes were removed. Right eyes were processed for electron microscopy (Poulsom, 1982). Left eyes were fixed in neutralized 10 y0 form01 saline containing marble chips, prior to the isolation (Kuwabara and Cogan, 1960) and staining with PAS/haematoxylin of their retinal vasculature. The right kidney was halved longitudinally through the hilum; half was processed for electron microscopy, the other half was fixed in neutralized form01 saline and paired stained sections (haematoxylin and eosin, PAS/haematoxylin) were prepared by the Histology Unit, Safety of Medicines Department, Pharmaceutical Division ICI plc. Assessment
of histological
changes
Code numbers were assigned to each eye by an independent person. Retinal vaseulatures were isolated, examined then assessed independently by R. P. and R. P. B.-H., in ignorance of their experimental treatments, for features indicative of retinopathy in diabetic rats (Papachristodoulou et al., 1976; Boot-Handford and Heath, 1980; Poulsom, 1982; see Introduction). Each assessor awarded each vasculature an integral score from 0 (no sign of pathological change) to 5 (severe retinopathy). The code was broken only when a third person confirmed that the scores were essentially in agreement. The ‘Retinal Pathology Score’ is the mean of the two independent scores. Similar precautions were taken for the assessment of pathological changes in the kidney sections as described elsewhere (Poulsom, 1982). Xtatistics Parametric observations (two-tailed)
results is given following
are expressed as the means with their standard errors. The number of in parentheses. These results are described as significant if P < 0.05 one-way analysis of variance or Student’s t test.
AR
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LONG-TER’M TABLE
DIABETIC
RATS
509
I
The effects of long-term (> 255 days) diabetes and ICI 105552 upon organ weights in the rat Organ Treatmentt
(g)*
Left kidney
Liver
CA CB DA DB
weight
21.1 f 1.32s
1+0+_0091()
18~o+oa3 19.5 + 1.42 17.1* 1.21
1?55*0~070 209+0127 2.00+0.25llj
SVCGS 1.90+_0.133 1.97 *0.079 1~60+0~391 1.95 + 0.467
* Mean+s.E.x. i CA = control dosed with ICI 105552 (n = 7), CB = control dosed with placebo diabetic dosed with ICI 105552 (n = 6), DB = diabetic dosed with placebo (n = 4). $ Seminal Vesicles with Coagulating Glands. 8 CA vs. CB, P c 0.05. I/ CA vs. CB, P = 0.010. 7 DB vs. CB, P < 001.
(TL = lo),
DA =
3. Results General Daily dosage with ICI 105552 for up to 373 days retarded the development of cataract and prevented the appearance of intraocular haemorrhage (Poulsom et al., 1982) and did not affect adversely the general health or behaviour of the rats. Due to the failing health of certain diabetic rats two rats per treatment were killed after 250 days of dosage. Three diabetic rats died suddenly after 320 days so the experiment was terminated with a mean duration of approximately 340 days. The terminal ( > 255 day) body weights (g) of diabetic rats were considerably less than those of controls: DA 386 f41.9 (6) vs. CA 589f14.7 (7), P < 0.0001; DB 414k28.7 (4) vs. CB 593 + 181 (lo), P < 60001. The inhibitor had no significant eEeet on body weight. Organ weights
(Table
I)
In control rats dosage with ICI 105552 increased both the mean liver weight (17 “&, P < 0.05) and mean left kidney weight (23 %, P = 0.010). The increases were insignificant in diabetic rats. The livers of diabetic rats were not significantly lighter than those of controls. The mean weight of left kidney was increased by diabetes; significantly in placebo-dosed rats (29%, P < O.Ol), non-significantly in ARI-dosed rats. Treatments did not alt,er the mean SVCG weight significantly. Terminal
blood glucose levels
The terminal fed-state glucose concentrations (m,n) were: CA 4.9f0.21 (9), CB 4.7kO.18 (ll), DA 17.9f4.90 (7), DB 18.7k4.06 (6). ICI 105552 had no significant effects; it was clearly not an hypoglycaemic agent. Gas-liquid
chromatographic
analyaea:
rats
surviving
> 240
days
ICI 105552 did not reduce the concentration of glucose significantly in the sciatic nerve, kidney (DA vs. DB, P > 0.6) or SVCG (DA vs. DB, P > 0.5). In the sciatic nerve (Table II) diabetes gave approximately a nine-fold increase in the concentration of sorbitol. Treatment with ICI 105552 reduced the accumulation by 86 %. The decrease was significant (DA vs. DB: P < 0.01). Similarly, the approximately eight-fold increase in the concentration of fructose was reduced by 69 oh (DA
510
R. POCLSOM TABLE
Biochemical
ET II
eflects of long-term ( > 240 days) diabetes and ICI 105552 in the sciatic nerve of the rat Concentration
Treatment? CA CB DA DB
BL.
GIUWXX (pwg) 1.63kO.143 1.65 + 0.078 103f215 $45 + 2.55
Sorbitol (nmol/g)
of substance* Fructose (nmol/g)
120f12 150* 15 310+50: 1300+ 190
650 + 49 790 * 72 2590+310f 6510+ 1150
Inositol MWg) 211&0.085 2~08~~100 2.33 f 0.095 2.05iO.218
* Mel/g wet weight : mean + S.E.M. t CA = control dosed with JCI 105552 (n = 9), CB = control dosed with placebo diabetic dosed with ICI 105552 (n = 5); DB = diabetic dosed with placebo (n = 3). $ DA vs. DB, P < 0.01.
TABLE
Biochemical
Concentration
CA CB DA DB
Glucose (k~ollg) 0.99 1.05 150 206
f + f f
0063 0.063 534 7.24
DA =
III
effects of long-term ( > 240 days) diabetes and ICI 105552 vesicles and coagulating glands of the rat
Treatment?
(n = ll),
Sorbitol (nmol/g) 200+14 2OOk15 310+45t 640 f 547
in the seminal
of substance* Fructose WWg) 2.47 kO.218 z43+@219 450 + 1.14s 6.50 It 0431
Inositol (pal/g) 226 + 0.79 20.6 + 090 13.1 f337// 16.9*30s
* Mel/g wet weight: mean+~.~.~. t CA = control dosed with ICI 105552 (n = 9), CB = control dosed with placebo diabetic dosed with ICI 105552 (n = 5), DB = diabetic dosed with placebo (n = 4). 1: DA vs. DB, P < 0005; DA vs. CB, P < 0.01. § DA vs. CB, P < 0.02. /I DA vs. CA, P < 0005. 1 DB vs. CB, P < 0001.
(n = ll),
DA =
vs. DB, P < 901). Neither diabetes nor the AR1 affected the concentration of inositol significantly. In the SVCG (Table III) diabetes increased the concentration of sorbitol three-fold (P < 0.0001); treatment with ICI 105552 reduced this accumulation by 75%. The decrease was significant (DA vs. DB, P < 9005) but the resulting level was still greater (55 %, P < 0.01) than the normal (CB) level. Fructose accumulated to 2.6-fold the normal concentration. The AR1 was able to reduce the increase by 49% but the concentration was still significantly greater (P < 902, 85%) than normal. Diabetes reduced the concentration of inositol in the SVCG considerably and variably; significantly in ARI-dosed rats (P < @005), non-significantly in placebo-dosed rats (P = 0.14). In the kidneys of diabetic rats (Table IV) variation in the concentration of sorbitol was so great that an effect of diabetes or the AR1 cannot be shown statistically.
AR
INHIBITION
IS
LQ?TG-TERM TABLE
Biochemical
CA CB DA DB
511
RATS
IV
e#ects of long-term (> 240 days) diabetes and ICI 105552 in the kidney of the rat Concentration
Treatment?
DIABETIC
Glucose WWg) 442 i 0.345 433ko.194 23.4 f 7.28 30.1+112
Sorbitol (nmol/g) 240 If: 38 3OQk36 280 & 39 570+ 198
of substance*Fructose (nmob) 370+53 370* 29 730 + 157 1920f863
Inositol (pwg) 606 504 538 6.51
* Mel/g wet weight: mean &- S.E.Y. t CA = control dosed with ICI 105552 (n = 5), CB = control dosed with placebo diabetic dosed with ICI 105552 (n = 4), DB = diabetic dosed with placebo (n = 4).
k + * f
0357 0.394 0.451 0.753
(n = ia),
DA =
Similarly, even though the mean concentration of fructose in the kidney was increased five-fold by diabetes alone and only two-fold in diabetics treated with ICI 105552: no significant effect can be shown. Neither diabetes nor the AR1 affected the concentration of inositol significantly.
Retinal
histoiogy
With. one or two exceptions, the severity of the pathological changes which developed in the retinas of SZ-diabetic rats was less than t.hat found in a previous experiment in this laboratory (Papachristodoulou et al., 1976). The distribution of retinal pathology scores (Fig. 1) shows the retinopathy to be worse than the control range in three of the diabetic rats. No effect of the duration of treatment was apparent but the fact that a range of scores was awarded to control rat retinas might in part be attributable to normal changes during aging (Glatt and Henkind, 1979). The retinal vasculature of one diabetic rat (No. 80 DA, dosed with ICI 105552 for 324 days) showed a severe retinopathy (Score 5) with extensive avascular regions, large networks of severely degenerated PAS + ve strands, many a,cellular and degenerating capillaries, a paucity of mural cells and formation of substantial shunt vessels (Fig. 2). This rat was severely diabetic, gaining only 13 g in weight, yet ICI 105552 prevented cataracts from developing beyond traces of opacity (Poulsom et al., 1982). Lesions were not apparent in the retinal vasculature of diabetic rat Xo. 32 DA (dosed with ICI 105552 for 336 days) despite the apparently constant presence of glucosuria over a nine-month period. Nowever, the rat gained 264 g in weight, Moderate to mild retinopathy was present in placebo-dosed diabetics No. 53 DB (score 3~5, dosed 336 days) and No. 35 DB (score 2.5, dosed 251 days). The eyes of both rats ha,d intraocular haemorrhages in vivo as did the eyes of most placebo-dosed diabetics, but never the eyes of ARI-treated diabetics (Poulsom et al., 1982). Renal
histology
The severity of pathological changes, especially tubular dilatation, varied within individual kidneys. Diabetes increased the severity of PAS +ve deposition in the mesangium, and to a lesser extent in the glomerular capillaries. Adenomas were present in rats injected with streptozotocin ; three of eight DA and two of six DB rats. There was no evidence that treatment with ICI 105552 had any effect on the renal pathology described.
512
4i
CA
CB
DA
D0
Treatment FIG. 1. The retinal pathology scores of normal and streptozotocin-diabetic 11 months with ICI 105552 01‘ a placebo. Abbreviation of treatment: ICI 105552, CB = control dosed with placebo, DA = diabetic dosed with dosed with placebo. Symbols with serif designate rats killed at approximately
rats treated for eight to CA = control dosed with ICI 105552, DB = diabetic 250 days of treatment.
FIG. 2. Part of the retinal vasculature of a streptozotocin-diabetic rat (NO. 80 DA) ICI 105552 for 324 days. One arteriolar branch (-) and many capillaries have degenerated, network of strongly PAS-positive strands. Certain vessels (s) may be shunts; they resemble emanate from an arteriole. Proliferation of endofhelial cell nuclei (e) has occurred. Approx.
dosed with forming a venules but x 80.
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513
microscopy
Preliminary studies of the retinas processed for electron microscopy revealed basement membrane thickening and other changes with @-diabetes but no effects of treatment with ICI 105552 were apparent (Poulsom, 1982). 4. Discussion The object of this investigation was to determine whether the oral treatment of SZ-diabetic rats for one year with an AR1 would prevent or ameliorate some or all of the complications which occur in long-standing diabetes. As difficulty has been experienced in maintaining rats with uncontrolled diabetes for six to 12 months, the dose of SZ administered iv. was initially set at 50 mg/kg body weight (Poulsom, 1982), which we have found in the past to induce a moderately severe diabetic state and to cause thedevelopment ofretinopathy (Papachristodoulouet al., 1976). Theretinopathy which ensued in the present study was not, however, as severe as that previously observed but nevertheless the daily treatment of SZ-diabetic rats for eight to 11 months with an AR1 did not affect in any way the degree of pathological change; in fact the most severely affected retinal vasculature was from an animal which had been treated with the AR1 for 324 days. The fact that the sorbitol pathway was still being inhibited after several months of treatment is shown by the levels of sorbitol found in the sciatic nerves of ARI-dosed and placebo-dosed diabetic rats where the AR1 treatment gave an 86 oh inhibition of the accumulation of sorbitol in diabetes both in the present investigation and in an earlier three-week study (Poulsom and Heath, 1983). In addition, the development of cataract was greatly retarded (Poulsom et al., 1982). On the other hand, there was no evidence that treatment with the AR1 had any effects on renal pathology (Poulsom, 1982). It would appear, therefore, that inhibition of the sorbitol pathway by a compound shown to be effective in vivo in the lens, sciatic nerve and seminal vesicles with coagulating glands of the rat is not readily demonstrated in the retina or kidney. In these two tissues of diabetic rats no statistically significa,nt lowering of sorbitol levels occurred with AR1 treatment even though the accumulations of sorbitol in the sciatic nerve, lens and seminal vesicles with coa,gulating glands were reduced by 86, 70 a,nd 55% respectively (Poulsom and Heath, 1983). The absence of an effect of this treatment upon the increased concentrations of sorbitol in the retinas of diabetic rats was confirmed in a subsequent short-term experiment (Poulsom, Mirrlees, Earl and Heath, 1983). It is possible that the activity of 1,(3,4-dichlorobenzyl)3-methyl-1,2-dihydro2-oxoquinol-4-ylacetic acid (sodium salt, ICI 105552), the AR1 used in this series of investigations, was not as effective against the retinal and renal enzymes as against those in the nerve, lens and seminal vesicles with coagula,ting glands since ARIs have been reported to give varying degrees of inhibition against aldose reductase isolated from different human tissues, such as lens and placenta,, and between species, such as rat and man (Kador, Kinoshita, Tung and Chylack, 1980). The implications of the results reported here therefore must be considered cautiously when applied to t,he possible beneficial effects which might be expected to occur on application of this type of chemotherapy to the diabetic complications of man.
514
R. POULSOM
ET
,4L
ACKNOWLEDGMENTS This work was supported by the Science Research Council through a C.A.S.E. Award to R. P. The authors wish to thank Dr D. C. N. Earl for his collaboration and advice, and for the gifts of ICI 105552 and placebo. We are grateful to MS H. V. Barclay for typing the manuscript. The streptozotocin was a gift from Upjohn, Kalamazoo, U.S.A. REFERENCES Babel,
J. and Leuenberger, P. (1974). A long term study on the ocular lesion in streptozotocin diabetic rats. Albrecht von Qraefes Arch. Klin. Exp. Ophthal. 189, 191-209. Beaumont, P., Hollows, F. C., Schofield, P. J., Williams, J. F. and Steinbeck, A. W. (1971). Hypothesis: growth hormone, sorbitol and diabetic capillary disease. The Lancet ii, 57!%31. Beyer-Mears, A. and Farnsworth, P. N. (1979). Diminished sugar cataractogenesis by quercetin. Exp. Eye Res. 28, 70916. Boot-Handford, R. and Heath, H. (1980). Identification of fructose as the retinopathic agent associated with ingestion of sucrose-rich diets in the rat. Metabolism 29, 1247-52. Cameron, D. P., Leuenberger, P., Amherdt, M., Mira, F., Orci, L. and Stauffacher, W. (1971). Microvascular lesions including retinal aneurysms in chronic experimental diabetes (streptozotocin). Eur. J. Clin. Invest. 1, 365 (Abstract). Fagius, J. and Jameson, S. (1981). Effects of aldose reductase inhibitor treatment in diabetic polyneuropathy - a clinical and neurophysiological study. J. Neurol. Neurosurg. Psychiat. 44, 991-1001. Glatt, H. J. and Henkind, P. (1979). Aging changes in the retinal capillary bed of the rat. Microvasc. Res. 18, l-17. Heath, H. and Hamlett, Y. C. (1976). The sorbitol pathway: effect of streptozotocin induced diabetes and the feeding of a sucrose-rich diet on glucose, sorbitol and fructose in the retina, blood and liver of rats. Diabetologia 12, 43-6. Judzewitsch, R., Jaspan, J. B., Pfeifer, M. A., Polonsky, K. S., Halar, E., Vukadinovic, C., Richton, S., Gabbay, K., Rubenstein, A. H. and Porte, D. (1981). Inhibition of aldose reductase improves motor nerve conduction velocity in diabetics. Diabetes 30 (Suppl. l), 30A. Kador, P. F., Kinoshita, J. H., Tung, W. H. and Chylack, L. T. (1980). Differences in the susceptibility of various aldose reductases to inhibition II. Invest. Ophthalmol. 19, 980-2. Kinoshita, J. H., Kador, P. and Datiles, M. (1981). Aldose reductase in diabetic catara,cts. J. Am. Med. Assoc. 246, 257-61. Kojima, K., Harada, T.; Hoshino, M. and Kojima, K. (1975). Electron microscopic observations on the retinal capillaries in streptozotocin-diabetic rat: with special reference to the pericyte of the capillary. Acta Sot. Ophthalmol. Jpn 79, 175&64. Kuwabara, T. and Cogan, D. G. (1960). Studies of retinal vascular patterns, part I normal architecture. Arch. Ophthalmol. 64, 904-l 1. Leuenberger, P. M. and Babel, J. (1978). Retinal microangiopathy in streptozotocin-diabetic rats. In Cellular and Biochemical Aspects of Diabetic Retinopathy (Eds Regnault, F. and Duhault, J.). INSERMSymposiumNo. 7. Pp. 41-56. Elsevier/North HollandBiomedical Press, Amsterdam. Papachristodoulou, D., Heath, H. and Kang, S. S. (1976). The development of setinopathy in sucrose-fed and streptozotocin-diabetic rats. Diabetologia 12, 367-74. Poulsom, R. (1982). Aldose reductase inhibition and experimental models of pathogenesis in diabetes. Ph.D. Thesis, University of London. Poulsom, R. and Heath, H. (1983). Inhibition of aldose reductase in five tissues of the streptozotocin-diabetic rat. Biochem. Pharmac. 32, 1495-99. Poulsom, R., Boot-Handford, R. P. and Heath, H. (1982). Some effects of aldose reductase inhibition upon the eyes of long-term streptozotocin-diabetic rats. Curr. Eye Res. 2, 351-55.
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Poulsom, R., Mirrlees, D. J., Earl, D. C. N. and Heath, H. (1983). The effects of an aldose reductase inhibitor upon the sorbitol pathway, fructose-l-phosphate and lactate in the retina and nerve of streptozotocin-diabetic rats. Exp. Eye Res. 36, 751-60. Varma, S. D., Mikuni, I. and Kinoshita, J. H. (1975). Flavonoids as inhibitors of lens aldose reductase. Scielzce 188, 121516.
The Effects tozotocin-diabetic
R.POULSOM*,
Depurtment
of Long-term ats with ~~b~bit~r R.
Treatment an Aldose
of Reductase
$.BooT-HA~DFORD~ANDH.HEATH
of Biochemical Pathology! University College School of Medicine, University Street, London WClE 6JJ (Received 7 March
1983 and accepted 12 April
1983)
To test the possible involvement of the sorbitol pathway in the pathogenesis of retinopathy in Long-term experimentally-diabetic rats, streptozotocin-diabetic and normal rats were dosed orally (50 mg/kg body weight daily) for ub to 373 days with an aldose reductase inhibitor (ICI 105552) or a placebo. Long-term treatment with ICI 105552 (1,(3,4-dichlorobenzyl)-3-methyl-l,2-dihydro-2-oxoquinol-4.ylacetic acid; sodium salt) markedly reduced the normal accumulations of sorbitol and fructose in the sciatic nerves (86 and 69 y0 reductions) and seminal vesicles with coagulating glands (75 and 49 % reductions). Thus. by these criteria, the inhibitor was as effective after several months of dosing as after three weeks. There was no evidence that treatment with this aldose reductase inhibitor had any protective effect against the development of pathological changes in the retina and kidney of these rats. Key words: aldose reductase inhibitor: ICI 105552 ; sorbitol ; fructose; inositol ; streptozotocindiabetes; diabetic retinopathy.
I. l[ntroduction Aldose reduetase (E.C. 1.1 1.21) is the first enzyme of the sorbitol pathway, which converts glucose into sorbitol and then into fructose. Abnormally increased activity of this pathway is considered to be a contributing factor in the pathogenesis of cataract (Kinoshita, Kador and Datiles, 1981) and polyneuropathy (Fagius and Jameson, 1984 ; Judzewitsch et al., 1981) in diabetes. An early experiment (Leuenberger and Babel, 1978) t’o test the hypothesis (Beaumont, Hollows, Schofield, Williams and Steinbeck, 1971; Heath and Hamlett, 1976) that the sorbitol pathway might be involved in the development of microvascular complications in diabetes was inconclusive since quercetin, the aldose reductase inhibitor (ARI) with which they treated their diabetic rats: had no demonstrable effects in vivo even though it was administered as a 5 o!0 (w/w) admixture to the diet. Quercetin has a moderate activity in vitro (Varma, Mikuni and Kinoshita, 1975) and rats (Beyer-Mears and measurable effects when given orally to galactosaemic Farnsworth, 1979). The present study was designed to test the hypothesis by treating streptozotocindiabetic (SZ-diabetic) rats with a new orally-active aldose reductase inhibitor, ICI 105552, for 12 months. The SZ-diabetic rat develops some of the retinal lesions of human diabetic retinopathy in six to 12 months. These changes include tortuosity and irregularity of capillaries, preferential loss of intra-mural pericytes (mural cells), acellular capillaries, increased PAS-positive deposits, thickening of capillary basement * Please address correspondence to Dr R. Poulsom, Departments of Visual Science Institute of Ophthalmology, Judd Street, London WClH QQS G.K. 7 Present address: Department of Medical Biochemistry, University of Manchester Stopford Building, Oxford Road, Manchester Ml3 QPT C.K.
001&4835/83/010507+10
SOS.OO/O
0
1983 Academic
and Pathology, Medical
Press Inc. (London)
School,
Limited
R. POULSOM
508
ET
AL.
membranes, strand formation and, infrequently, fusiform or saccular microaneurysms (Cameron et al., 1971; Babel and Leuenberger, 1974; Kojima, Harada, Hoshino and Kojima, 1975; Papachristodoulou, Heath and Kang, 1976; Leuenberger and Babel, 1978; Boot-Handford and Heath, 1980). It was anticipated that during the course of the experiment the inhibitor would prevent or retard cataractogenesis; this was so and these results have been reported and discussed previously (Poulsom, Boot-Handford and Heath, 1982). In order to demonstrate that the aldose reductase inhibitor retained its effectiveness after many months of dosing the concentrations of glucose, sorbitol and fructose were measured in tissues known to possess the sorbitol pathway and these results were compared with those from a short-term study (Poulsom and Heath, 1983). 2. Materials Animals
and
Methods
and treatments
These details have been reported previously (Poulsom et al., 1982). Briefly, male (299 g) Wistar rats, housed in wire-bottomed cages with a standard diet (MRC 4lB) and water available ad libitum were assigned randomly to one of the four treatments; diabetic dosed with ICI 105552 (designated as treatment DA), diabetic dosed with placebo (DB), control dosed with ICI 105552 (CA), control dosed with placebo (CB). Diabetes was induced with streptozotocin (i.v.) and was confirmed if glucosuria and a fed-state blood glucose level > 16-6 mM were present one week later; these criteria were used periodically to confirm the persistence of diabetes. When necessary, further doses of the diabetogen were given (Poulsom, 1982). One week after the induction of diabetes once-daily dosage with the enzyme inhibitor or placebo as appropriate was commenced. Rats, originally 12 per treatment, were dosed at 50 mg/kg body weight by gastric intubation with a 25 g/l solution in deionized distilled water for up to 373 days. Preparation
of tissues
Two rats per day were anaesthetized deeply (Sagatal, May & Baker; 6&72 mg/kg body weight) before noon and at least 45 min after their last oral dose of ICI 105552 or placebo. Enzymatic or gas-liquid chromatographic methods (Poulsom and Heath, 1983) were used to assay glucose in tail vein blood and sugars and polyols in samples of the left kidney, the sciatic nerves and the seminal vesicles with attached coagnlating glands (SVCG). The eyes were removed. Right eyes were processed for electron microscopy (Poulsom, 1982). Left eyes were fixed in neutralized 10 y0 form01 saline containing marble chips, prior to the isolation (Kuwabara and Cogan, 1960) and staining with PAS/haematoxylin of their retinal vasculature. The right kidney was halved longitudinally through the hilum; half was processed for electron microscopy, the other half was fixed in neutralized form01 saline and paired stained sections (haematoxylin and eosin, PAS/haematoxylin) were prepared by the Histology Unit, Safety of Medicines Department, Pharmaceutical Division ICI plc. Assessment
of histological
changes
Code numbers were assigned to each eye by an independent person. Retinal vaseulatures were isolated, examined then assessed independently by R. P. and R. P. B.-H., in ignorance of their experimental treatments, for features indicative of retinopathy in diabetic rats (Papachristodoulou et al., 1976; Boot-Handford and Heath, 1980; Poulsom, 1982; see Introduction). Each assessor awarded each vasculature an integral score from 0 (no sign of pathological change) to 5 (severe retinopathy). The code was broken only when a third person confirmed that the scores were essentially in agreement. The ‘Retinal Pathology Score’ is the mean of the two independent scores. Similar precautions were taken for the assessment of pathological changes in the kidney sections as described elsewhere (Poulsom, 1982). Xtatistics Parametric observations (two-tailed)
results is given following
are expressed as the means with their standard errors. The number of in parentheses. These results are described as significant if P < 0.05 one-way analysis of variance or Student’s t test.
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509
I
The effects of long-term (> 255 days) diabetes and ICI 105552 upon organ weights in the rat Organ Treatmentt
(g)*
Left kidney
Liver
CA CB DA DB
weight
21.1 f 1.32s
1+0+_0091()
18~o+oa3 19.5 + 1.42 17.1* 1.21
1?55*0~070 209+0127 2.00+0.25llj
SVCGS 1.90+_0.133 1.97 *0.079 1~60+0~391 1.95 + 0.467
* Mean+s.E.x. i CA = control dosed with ICI 105552 (n = 7), CB = control dosed with placebo diabetic dosed with ICI 105552 (n = 6), DB = diabetic dosed with placebo (n = 4). $ Seminal Vesicles with Coagulating Glands. 8 CA vs. CB, P c 0.05. I/ CA vs. CB, P = 0.010. 7 DB vs. CB, P < 001.
(TL = lo),
DA =
3. Results General Daily dosage with ICI 105552 for up to 373 days retarded the development of cataract and prevented the appearance of intraocular haemorrhage (Poulsom et al., 1982) and did not affect adversely the general health or behaviour of the rats. Due to the failing health of certain diabetic rats two rats per treatment were killed after 250 days of dosage. Three diabetic rats died suddenly after 320 days so the experiment was terminated with a mean duration of approximately 340 days. The terminal ( > 255 day) body weights (g) of diabetic rats were considerably less than those of controls: DA 386 f41.9 (6) vs. CA 589f14.7 (7), P < 0.0001; DB 414k28.7 (4) vs. CB 593 + 181 (lo), P < 60001. The inhibitor had no significant eEeet on body weight. Organ weights
(Table
I)
In control rats dosage with ICI 105552 increased both the mean liver weight (17 “&, P < 0.05) and mean left kidney weight (23 %, P = 0.010). The increases were insignificant in diabetic rats. The livers of diabetic rats were not significantly lighter than those of controls. The mean weight of left kidney was increased by diabetes; significantly in placebo-dosed rats (29%, P < O.Ol), non-significantly in ARI-dosed rats. Treatments did not alt,er the mean SVCG weight significantly. Terminal
blood glucose levels
The terminal fed-state glucose concentrations (m,n) were: CA 4.9f0.21 (9), CB 4.7kO.18 (ll), DA 17.9f4.90 (7), DB 18.7k4.06 (6). ICI 105552 had no significant effects; it was clearly not an hypoglycaemic agent. Gas-liquid
chromatographic
analyaea:
rats
surviving
> 240
days
ICI 105552 did not reduce the concentration of glucose significantly in the sciatic nerve, kidney (DA vs. DB, P > 0.6) or SVCG (DA vs. DB, P > 0.5). In the sciatic nerve (Table II) diabetes gave approximately a nine-fold increase in the concentration of sorbitol. Treatment with ICI 105552 reduced the accumulation by 86 %. The decrease was significant (DA vs. DB: P < 0.01). Similarly, the approximately eight-fold increase in the concentration of fructose was reduced by 69 oh (DA
510
R. POCLSOM TABLE
Biochemical
ET II
eflects of long-term ( > 240 days) diabetes and ICI 105552 in the sciatic nerve of the rat Concentration
Treatment? CA CB DA DB
BL.
GIUWXX (pwg) 1.63kO.143 1.65 + 0.078 103f215 $45 + 2.55
Sorbitol (nmol/g)
of substance* Fructose (nmol/g)
120f12 150* 15 310+50: 1300+ 190
650 + 49 790 * 72 2590+310f 6510+ 1150
Inositol MWg) 211&0.085 2~08~~100 2.33 f 0.095 2.05iO.218
* Mel/g wet weight : mean + S.E.M. t CA = control dosed with JCI 105552 (n = 9), CB = control dosed with placebo diabetic dosed with ICI 105552 (n = 5); DB = diabetic dosed with placebo (n = 3). $ DA vs. DB, P < 0.01.
TABLE
Biochemical
Concentration
CA CB DA DB
Glucose (k~ollg) 0.99 1.05 150 206
f + f f
0063 0.063 534 7.24
DA =
III
effects of long-term ( > 240 days) diabetes and ICI 105552 vesicles and coagulating glands of the rat
Treatment?
(n = ll),
Sorbitol (nmol/g) 200+14 2OOk15 310+45t 640 f 547
in the seminal
of substance* Fructose WWg) 2.47 kO.218 z43+@219 450 + 1.14s 6.50 It 0431
Inositol (pal/g) 226 + 0.79 20.6 + 090 13.1 f337// 16.9*30s
* Mel/g wet weight: mean+~.~.~. t CA = control dosed with ICI 105552 (n = 9), CB = control dosed with placebo diabetic dosed with ICI 105552 (n = 5), DB = diabetic dosed with placebo (n = 4). 1: DA vs. DB, P < 0005; DA vs. CB, P < 0.01. § DA vs. CB, P < 0.02. /I DA vs. CA, P < 0005. 1 DB vs. CB, P < 0001.
(n = ll),
DA =
vs. DB, P < 901). Neither diabetes nor the AR1 affected the concentration of inositol significantly. In the SVCG (Table III) diabetes increased the concentration of sorbitol three-fold (P < 0.0001); treatment with ICI 105552 reduced this accumulation by 75%. The decrease was significant (DA vs. DB, P < 9005) but the resulting level was still greater (55 %, P < 0.01) than the normal (CB) level. Fructose accumulated to 2.6-fold the normal concentration. The AR1 was able to reduce the increase by 49% but the concentration was still significantly greater (P < 902, 85%) than normal. Diabetes reduced the concentration of inositol in the SVCG considerably and variably; significantly in ARI-dosed rats (P < @005), non-significantly in placebo-dosed rats (P = 0.14). In the kidneys of diabetic rats (Table IV) variation in the concentration of sorbitol was so great that an effect of diabetes or the AR1 cannot be shown statistically.
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Biochemical
CA CB DA DB
511
RATS
IV
e#ects of long-term (> 240 days) diabetes and ICI 105552 in the kidney of the rat Concentration
Treatment?
DIABETIC
Glucose WWg) 442 i 0.345 433ko.194 23.4 f 7.28 30.1+112
Sorbitol (nmol/g) 240 If: 38 3OQk36 280 & 39 570+ 198
of substance*Fructose (nmob) 370+53 370* 29 730 + 157 1920f863
Inositol (pwg) 606 504 538 6.51
* Mel/g wet weight: mean &- S.E.Y. t CA = control dosed with ICI 105552 (n = 5), CB = control dosed with placebo diabetic dosed with ICI 105552 (n = 4), DB = diabetic dosed with placebo (n = 4).
k + * f
0357 0.394 0.451 0.753
(n = ia),
DA =
Similarly, even though the mean concentration of fructose in the kidney was increased five-fold by diabetes alone and only two-fold in diabetics treated with ICI 105552: no significant effect can be shown. Neither diabetes nor the AR1 affected the concentration of inositol significantly.
Retinal
histoiogy
With. one or two exceptions, the severity of the pathological changes which developed in the retinas of SZ-diabetic rats was less than t.hat found in a previous experiment in this laboratory (Papachristodoulou et al., 1976). The distribution of retinal pathology scores (Fig. 1) shows the retinopathy to be worse than the control range in three of the diabetic rats. No effect of the duration of treatment was apparent but the fact that a range of scores was awarded to control rat retinas might in part be attributable to normal changes during aging (Glatt and Henkind, 1979). The retinal vasculature of one diabetic rat (No. 80 DA, dosed with ICI 105552 for 324 days) showed a severe retinopathy (Score 5) with extensive avascular regions, large networks of severely degenerated PAS + ve strands, many a,cellular and degenerating capillaries, a paucity of mural cells and formation of substantial shunt vessels (Fig. 2). This rat was severely diabetic, gaining only 13 g in weight, yet ICI 105552 prevented cataracts from developing beyond traces of opacity (Poulsom et al., 1982). Lesions were not apparent in the retinal vasculature of diabetic rat Xo. 32 DA (dosed with ICI 105552 for 336 days) despite the apparently constant presence of glucosuria over a nine-month period. Nowever, the rat gained 264 g in weight, Moderate to mild retinopathy was present in placebo-dosed diabetics No. 53 DB (score 3~5, dosed 336 days) and No. 35 DB (score 2.5, dosed 251 days). The eyes of both rats ha,d intraocular haemorrhages in vivo as did the eyes of most placebo-dosed diabetics, but never the eyes of ARI-treated diabetics (Poulsom et al., 1982). Renal
histology
The severity of pathological changes, especially tubular dilatation, varied within individual kidneys. Diabetes increased the severity of PAS +ve deposition in the mesangium, and to a lesser extent in the glomerular capillaries. Adenomas were present in rats injected with streptozotocin ; three of eight DA and two of six DB rats. There was no evidence that treatment with ICI 105552 had any effect on the renal pathology described.
512
4i
CA
CB
DA
D0
Treatment FIG. 1. The retinal pathology scores of normal and streptozotocin-diabetic 11 months with ICI 105552 01‘ a placebo. Abbreviation of treatment: ICI 105552, CB = control dosed with placebo, DA = diabetic dosed with dosed with placebo. Symbols with serif designate rats killed at approximately
rats treated for eight to CA = control dosed with ICI 105552, DB = diabetic 250 days of treatment.
FIG. 2. Part of the retinal vasculature of a streptozotocin-diabetic rat (NO. 80 DA) ICI 105552 for 324 days. One arteriolar branch (-) and many capillaries have degenerated, network of strongly PAS-positive strands. Certain vessels (s) may be shunts; they resemble emanate from an arteriole. Proliferation of endofhelial cell nuclei (e) has occurred. Approx.
dosed with forming a venules but x 80.
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513
microscopy
Preliminary studies of the retinas processed for electron microscopy revealed basement membrane thickening and other changes with @-diabetes but no effects of treatment with ICI 105552 were apparent (Poulsom, 1982). 4. Discussion The object of this investigation was to determine whether the oral treatment of SZ-diabetic rats for one year with an AR1 would prevent or ameliorate some or all of the complications which occur in long-standing diabetes. As difficulty has been experienced in maintaining rats with uncontrolled diabetes for six to 12 months, the dose of SZ administered iv. was initially set at 50 mg/kg body weight (Poulsom, 1982), which we have found in the past to induce a moderately severe diabetic state and to cause thedevelopment ofretinopathy (Papachristodoulouet al., 1976). Theretinopathy which ensued in the present study was not, however, as severe as that previously observed but nevertheless the daily treatment of SZ-diabetic rats for eight to 11 months with an AR1 did not affect in any way the degree of pathological change; in fact the most severely affected retinal vasculature was from an animal which had been treated with the AR1 for 324 days. The fact that the sorbitol pathway was still being inhibited after several months of treatment is shown by the levels of sorbitol found in the sciatic nerves of ARI-dosed and placebo-dosed diabetic rats where the AR1 treatment gave an 86 oh inhibition of the accumulation of sorbitol in diabetes both in the present investigation and in an earlier three-week study (Poulsom and Heath, 1983). In addition, the development of cataract was greatly retarded (Poulsom et al., 1982). On the other hand, there was no evidence that treatment with the AR1 had any effects on renal pathology (Poulsom, 1982). It would appear, therefore, that inhibition of the sorbitol pathway by a compound shown to be effective in vivo in the lens, sciatic nerve and seminal vesicles with coagulating glands of the rat is not readily demonstrated in the retina or kidney. In these two tissues of diabetic rats no statistically significa,nt lowering of sorbitol levels occurred with AR1 treatment even though the accumulations of sorbitol in the sciatic nerve, lens and seminal vesicles with coa,gulating glands were reduced by 86, 70 a,nd 55% respectively (Poulsom and Heath, 1983). The absence of an effect of this treatment upon the increased concentrations of sorbitol in the retinas of diabetic rats was confirmed in a subsequent short-term experiment (Poulsom, Mirrlees, Earl and Heath, 1983). It is possible that the activity of 1,(3,4-dichlorobenzyl)3-methyl-1,2-dihydro2-oxoquinol-4-ylacetic acid (sodium salt, ICI 105552), the AR1 used in this series of investigations, was not as effective against the retinal and renal enzymes as against those in the nerve, lens and seminal vesicles with coagula,ting glands since ARIs have been reported to give varying degrees of inhibition against aldose reductase isolated from different human tissues, such as lens and placenta,, and between species, such as rat and man (Kador, Kinoshita, Tung and Chylack, 1980). The implications of the results reported here therefore must be considered cautiously when applied to t,he possible beneficial effects which might be expected to occur on application of this type of chemotherapy to the diabetic complications of man.
514
R. POULSOM
ET
,4L
ACKNOWLEDGMENTS This work was supported by the Science Research Council through a C.A.S.E. Award to R. P. The authors wish to thank Dr D. C. N. Earl for his collaboration and advice, and for the gifts of ICI 105552 and placebo. We are grateful to MS H. V. Barclay for typing the manuscript. The streptozotocin was a gift from Upjohn, Kalamazoo, U.S.A. REFERENCES Babel,
J. and Leuenberger, P. (1974). A long term study on the ocular lesion in streptozotocin diabetic rats. Albrecht von Qraefes Arch. Klin. Exp. Ophthal. 189, 191-209. Beaumont, P., Hollows, F. C., Schofield, P. J., Williams, J. F. and Steinbeck, A. W. (1971). Hypothesis: growth hormone, sorbitol and diabetic capillary disease. The Lancet ii, 57!%31. Beyer-Mears, A. and Farnsworth, P. N. (1979). Diminished sugar cataractogenesis by quercetin. Exp. Eye Res. 28, 70916. Boot-Handford, R. and Heath, H. (1980). Identification of fructose as the retinopathic agent associated with ingestion of sucrose-rich diets in the rat. Metabolism 29, 1247-52. Cameron, D. P., Leuenberger, P., Amherdt, M., Mira, F., Orci, L. and Stauffacher, W. (1971). Microvascular lesions including retinal aneurysms in chronic experimental diabetes (streptozotocin). Eur. J. Clin. Invest. 1, 365 (Abstract). Fagius, J. and Jameson, S. (1981). Effects of aldose reductase inhibitor treatment in diabetic polyneuropathy - a clinical and neurophysiological study. J. Neurol. Neurosurg. Psychiat. 44, 991-1001. Glatt, H. J. and Henkind, P. (1979). Aging changes in the retinal capillary bed of the rat. Microvasc. Res. 18, l-17. Heath, H. and Hamlett, Y. C. (1976). The sorbitol pathway: effect of streptozotocin induced diabetes and the feeding of a sucrose-rich diet on glucose, sorbitol and fructose in the retina, blood and liver of rats. Diabetologia 12, 43-6. Judzewitsch, R., Jaspan, J. B., Pfeifer, M. A., Polonsky, K. S., Halar, E., Vukadinovic, C., Richton, S., Gabbay, K., Rubenstein, A. H. and Porte, D. (1981). Inhibition of aldose reductase improves motor nerve conduction velocity in diabetics. Diabetes 30 (Suppl. l), 30A. Kador, P. F., Kinoshita, J. H., Tung, W. H. and Chylack, L. T. (1980). Differences in the susceptibility of various aldose reductases to inhibition II. Invest. Ophthalmol. 19, 980-2. Kinoshita, J. H., Kador, P. and Datiles, M. (1981). Aldose reductase in diabetic catara,cts. J. Am. Med. Assoc. 246, 257-61. Kojima, K., Harada, T.; Hoshino, M. and Kojima, K. (1975). Electron microscopic observations on the retinal capillaries in streptozotocin-diabetic rat: with special reference to the pericyte of the capillary. Acta Sot. Ophthalmol. Jpn 79, 175&64. Kuwabara, T. and Cogan, D. G. (1960). Studies of retinal vascular patterns, part I normal architecture. Arch. Ophthalmol. 64, 904-l 1. Leuenberger, P. M. and Babel, J. (1978). Retinal microangiopathy in streptozotocin-diabetic rats. In Cellular and Biochemical Aspects of Diabetic Retinopathy (Eds Regnault, F. and Duhault, J.). INSERMSymposiumNo. 7. Pp. 41-56. Elsevier/North HollandBiomedical Press, Amsterdam. Papachristodoulou, D., Heath, H. and Kang, S. S. (1976). The development of setinopathy in sucrose-fed and streptozotocin-diabetic rats. Diabetologia 12, 367-74. Poulsom, R. (1982). Aldose reductase inhibition and experimental models of pathogenesis in diabetes. Ph.D. Thesis, University of London. Poulsom, R. and Heath, H. (1983). Inhibition of aldose reductase in five tissues of the streptozotocin-diabetic rat. Biochem. Pharmac. 32, 1495-99. Poulsom, R., Boot-Handford, R. P. and Heath, H. (1982). Some effects of aldose reductase inhibition upon the eyes of long-term streptozotocin-diabetic rats. Curr. Eye Res. 2, 351-55.
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Poulsom, R., Mirrlees, D. J., Earl, D. C. N. and Heath, H. (1983). The effects of an aldose reductase inhibitor upon the sorbitol pathway, fructose-l-phosphate and lactate in the retina and nerve of streptozotocin-diabetic rats. Exp. Eye Res. 36, 751-60. Varma, S. D., Mikuni, I. and Kinoshita, J. H. (1975). Flavonoids as inhibitors of lens aldose reductase. Scielzce 188, 121516.