Ganglioside Treatment of Streptozotocin-Diabetic Rats Prevents Defective Axonal Transport of 6-Phosphofructokinase Activity

Journal of Neurochemis~ry Raven Press, Ltd., New York 0 1988 International Society for Neurochemistry

Ganglioside Treatment of Streptozotocin-Diabetic Rats Prevents Defective Axonal Transport of 6-Phosphofructokinase Activity N. A. Calcutt, D. R. Tomlinson, and G. B. Willars Department of Physiology and Pharmucologj-, Medical School, Qireen 's Medical Centre, Nottingham, England

Abstract: This study measured axonal transport of 6-phosphofmctokinase (PFK) and aldolase activities in the sciatic nerves of rats with short-term streptozotocin-induced diabetes. The diabetic rats showed deficits in anterograde (69% of controls; p < 0.001) and retrograde (3370 of controls: p < 0.01) accumulations of PFK activity as well as its content per unit length of unconstricted sciatic nerve (86% of controls; p < 0.05). There were no accumulation deficits in aldolase activity in the nerves of the diabetic rats, although the activity per unit length of unconstricted nerve was deficient (81% of controls; p < 0.05). Treatment ofdiabetic rats with mixed bovine brain gangliosides (10 rng/kg of body weight/day, i.p.) did not affect the deficit in PFK activity in unconstricted nerve (84% of ganglioside-treated controls; p

< O.Ol), but all the other defects in enzyme activities were prevented completely. The diabetic rats also showed a reduction of 7% (p < 0.01) in sciatic nerve dry weight per unit length, which was prevented by ganglioside treatment. In contrast, the reduced motor nerve conduction velocity, accumulation of polyol pathway metabolites, and depletion of mjwinositol, characteristic of untreated short-term diabetes. were unaffected by ganglioside treatment. Key Words: Aldolase-Axonal transport-DiabetesGangliosides-Phosphofructokinase. Calcutt N. A. et al. Ganglioside treatment of streptozotocin-diabetic rats prevents defective axonal transport of 6-phosphofructokinase activity. J. Neurochem. 50, 1478-1483 (1988).

The axon and terminals of neurones are dependent on the supply of macromolecules synthesised in the perikaryon for their continued maintenance and function. Delivery of materials is achieved by processes collectively termed anterograde axonal transport. Retrograde axonal transport also occurs and represents both the return of surplus material and the delivery of exogenous substances to the cell body. thereby potentially regulating protein synthesis (Grafstein and Forman, 1980). Defects in anterograde and retrograde axonal transport have been reported in several neuropathies, including experimental diabetic neuropathy, and precede structural damage to the neurone (for reviews, see Sidenius, 1982: Tomlinson and Mayer, 1984; Jakobsen et al., 1986). Interventions that prevent or attenuate the development of defects of axonal transport in experimental diabetes, therefore, provide insight into the pathogenesis of diabetic neuropathies and potential therapeu-

tic or prophylactic measures against their clinical manifestation. Axonal transport of two axoplasmic enzyme activities has been studied in detail in streptozotocininduced diabetic rats in our laboratory: acetylCoA:choline 0-acetyltransferase (EC 2.3.1.6; choline acetyltransferase) and ATP:D-fructose-6-phosphate 1-phosphotransferase [EC 2.7.1.1 1; 6-phosphofructokinase (PFK)]. Both activities are moved in the slow component of anterograde axonal transport in the sciatic nerve of the rat (Saunders et al., 1973; Willars et al., 19876). Both activities show deficits in accumulation proximal to 24-h constrictions of the sciatic nerves in rats with diabetes of 3-4 weeks in duration (Schmidt et al., 1975; Tomlinson et al., 1987). However, the accumulation deficits for these two enzyme activities arise from different aetiologies. The deficit in accumulation of choline acetyltransferase is prevented by treatment with a range of aldose reductase

Received May 4, 1987; revised manuscript received November 17, 1987; accepted November 20, 1987. Address correspondence and reprint requests to Dr. N. A. Cal-

Abbreviations used: MNCV, motor nerve conduction velocity; PFK, 6-phosphofructokinase.

cutt at Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham N G 7 2UH. U.K.

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GANGLIOSIDES IN DIABETIC RATS inhibitors (Tomlinson et al., 1982, 1984; Mayer and Tomlinson, 1983), whereas the deficit in accumulation of PFK is completely resistant to such treatment (Tomlinson et al., 1987; Willars et al., 1987a). It, therefore, appears that the defect in axonal transport of choline acetyltransferase activity is related to accumulation of polyol pathway metabolites and myoinositol depletion (Mayer and Tomlinson, 1983). Possible mechanisms are reviewed elsewhere (Greene et al., 1985). The present study was designed to begin a n examination of the possible origins of the defect in axonal transport of PFK activity. Studies using genetically diabetic mice have demonstrated that neuropathic manifestations of chronic diabetes, including changes in axonal morphometry and decreased motor nerve conduction velocity (MNCV), may be prevented by treatment with bovine brain gangliosides (Norido et al., 1984). Gangliosides are glycosphingolipids that contain sialic acid and are primarily membrane components, particularly abundant in neural tissue of the CNS. Purified ganglioside preparations have been demonstrated to have b o t h neuronotrophic and neuritogenic properties (Ledeen, 1984), as well as the ability to protect and stabilize membrane function against challenge (Vyskocil et al., 1985; Janigro et al., 1984; Bianchi et al., 1986). Ganglioside treatment of diabetic rats has recently been shown to prevent the development of accumulation defects of acetylcholine acetylhydrolase (EC 3.1.1.7; acetylcholinesterase) molecular forms that travel in both slow and fast components of axonal transport (Marini et al., 1986). We, therefore, decided t o investigate whether ganglioside treatment of acutely diabetic rats had any influence on the defect in PFK activity transport. Because previous studies had demonstrated, in the sciatic nerves of diabetic rats, a deficit in the accumulation at a ligature of one glycolytic enzyme-PFK activity-measurements for the present study were widened to encompass measurements of the activity of a second glycolytic enzyme-D-fructose- 1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase (EC 4.1.2.13; aldolase). As a basis for comparison with other studies referred to above, MNCV was also measured at the end of the diabetes/treatment protocol.

MATERIALS AND METHODS Groups and treatments Male Wistar rats weighing -280-300 g were randomly assigned to four groups and fasted overnight. Animals from two of the four groups were given a single intraperitoneal injection of streptozotocin (50 mg/kg in saline) to induce diabetes mellitus. Two days after injection of streptozotocin, the blood glucose concentration in the tail vein was determined using a glucose oxidase strip-operated reflectance meter (Reflolux; Boehringer Corp., London, U.K.). Animals with blood glucose concentrations of 1 cm proximal or distal to the point of ligation. Details for PFK are given elsewhere (Willars et al., 19876). Consequently, net anterograde accumulation was calculated as the activity in the two 5-mm segments proximal to the ligature minus twice the mean background activity. Net retrograde accumulation was calculated as the activity in the two 5-mm distal segments minus twice the mean background activity.

Quantification of nerve monosaccharides, polyols, and water content Remaining portions of the unconstricted right sciatic nerve were measured, blotted three times on filter paper. and then weighed. Nerves were then freeze-dried and reweighed. Nerve dry weight per unit length was calculated in milligrams per centimeter, and water content was expressed as milligrams of water per milligram dry weight of nerve. The sugar and polyol contents of these portions of nerve were then determined by GLC of their extracted trimethylsilyl derivatives using an SE52 packed column and flame ionization detection, with a-methyl mannoside as an internal standard (for the full method, see Tomlinson et al.. 1984).

Statistical analysis For all measurements, the untreated diabetic rats were compared with untreated controls, and ganglioside-treated diabetic rats were compared with ganglioside-treated controls. Unpaired t tests were used.

RESULTS Body weight, plasma glucose, and MNCV Both groups of diabetic animals lost weight compared with control animals (Table 1). Plasma glucose content was significantly elevated in both the untreated and ganglioside-treated diabetic rats when compared with the appropriate control group (both p < 0.001). Both groups of diabetic rats had significantly lower MNCV values than their respective conJ Nrirrochem.. V d 50, No. 5 , I988

TABLE I . Final body weight, plasma glucose level at death, and MNCV in control and streptozotocin-induced diabetic rats with and without nandioside treatment (g)

Plasma glucose (mmol/L)

MNCV Ws)

Untreated Controls (9) Diabetics (9)

324 ? 5 231 10

*

7.0 k 0.3 39.8 rt 2.7

52.0 f 0.9“ 43.8 f 0.8

Ganglioside-treated Controls (9) Diabetics (8)

323 ? 8 224 ? 1 1

6.9 k 0.3 35.7 1.7

50.2 f 0.7” 43.2 + 0.7

Body weight

*

Data are mean t SEM values; numbers of rats are given in parentheses. Levels of significance (derived by unpaired t tests) compared with diabetic rats are as follows: “p < 0.00 1. Massive differences were not tested.

trol groups (both p < 0.001). Thus, there was no indication of an effect of ganglioside treatment on the MNCV deficit of short-term diabetes. Enzyme activity and accumulation PFK activity in the unconstricted right sciatic nerve, expressed per mm of nerve, was significantly reduced in untreated diabetic rats when compared with untreated control rats (p < 0.05; Table 2). The accumulation of PFK activity in the nerves of untreated diabetic rats was 69% of controls proximal to the ligature (p < 0.001) and 33% of controls distal to the ligature (p < 0.0 1). Ganglioside-treated diabetic rats also showed a reduction in background PFK activity when compared with ganglioside-treated control animals (p < 0.01). However, there were no significant differences in either proximal or distal accumulation of PFK activity between diabetic or control rats treated with ganglioside. Background aldolase activity was significantly reduced in untreated diabetic rats compared with untreated control animals (p < 0.05; Table 2). No significant reduction in background aldolase activity was detected in ganglioside-treated diabetic animals when compared with ganglioside-treated controls. There were no significant changes in either proximal or distal accumulations of aldolase activity between any of the experimental groups. Sciatic nerve water, monosaccharide, and polyol content These data are presented in Table 3. No significant differences were observed in nerve water content, expressed as milligrams of water per milligram of dry nerve, between any of the groups. Untreated diabetic rats exhibited reduced nerve dry weight when compared with untreated controls (p < 0.01). No such difference was apparent in ganglioside-treated diabetic rats, with values not different from those of ganglioside-treated control rats. The contents of glucose, sorbitol, and fructose were all significantly elevated in

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TABLE 2. Activities of PFK and aldolase in unconstricted sciatic nerve (background) and accumulated proximal and distal to 24-h constrictions applied to the contralateral sciatic nerve in four groups of rats PFK activity

Aldolase activity

Background (per mm of nerve)

Proximal accumulation

Distal accumulation

Background (per mm of nerve)

Proximal accumulation

Distal accumulation

Untreated Controls (9) Diabetics (9)

2.16 f 0.08" 1.84 f 0.12

6.60 f 0.32' 3.91 f 0.57

2.12 f 0.33' 0.71 f 0.24

0.584 f 0.033" 0.474 f 0.020

2.85 f 0.47 2.49 f 0.26

1.56 k 0.31 1.71 rt_ 0.41

Ganglioside-treated Controls (9) Diabetics (8)

2.26 ? 0.06' 1.90 f 0.08

6.23 f 0.63 6.18 f 0.51

1.48 f 0.34 1.81 f 0.35

0.579 f 0.028 0.540 f 0.025

2.52 f 0.30 2.47 f 0.24

1.57 f 0.37 1.30 f 0.36

Enzyme activities are expressed as nmol of substrate used/min. Data are mean f SEM values; numbers of rats are given in parentheses. Levels of significance (derived by unpaired t tests) compared with diabetic rats are as follows: "p < 0.05, 'p < 0.001, 'p < 0.01,

the nerves of both untreated and ganglioside-treated diabetic rats when compared with the appropriate control group (all p < 0.01). Nerve myo-inositol content was similarly significantly reduced in both diabetic groups compared with their matched control groups (both p < 0.001). Thus, ganglioside treatment was without effect on the monosaccharides and polyols assayed.

DISCUSSION Other studies have explored the relationships among polyol pathway metabolites, myo-inositol, and MNCV (see, for example, Greene et al., 1975; Tomlinson et al., 1982; Finegold et al., 1983; Mayer and Tomlinson, 1983). Ganglioside treatment, at the dose and duration used in this study, altered none of these variables in diabetic rats. Thus, it appears disorders that are related to exaggerated polyol pathway flux and that may be prevented by aldose reductase inhibitors are not affected by gangliosides. The lack of prevention of the MNCV defect by gangliosides, at least within the time frame studied, is consistent with the findings of a previous study (Norido et al., 1984). That study used genetically diabetic

mice and showed that gangliosides protected against MNCV defects developing in chronically diabetic animals but were ineffective against acute conduction defects. The former may develop as a consequence of structural breakdown of the axonal endoskeleton, whereas the latter are more likely to be a result of acute biochemical aberrations (Sidenius, 1982), probably related to the polyol pathway. The major effect of gangliosides in the present study was to prevent the accumulation deficits in PFK activity in constricted sciatic nerves of diabetic rats. We have previously characterised the axonal transport of PFK activity in the sciatic nerves of Wistar rats using single and double ligation studies (Willars et al., 19876). The increase in enzyme activity proximal to a ligature was linear for at least 48 h. Distal to a ligature, linearity of accumulation persisted for 24 h. Double ligation experiments showed no redistribution of enzyme activity in the portion of nerve isolated between the ligatures during a 24-h period. Thus, calculation of a mobile fraction of PFK activity is not possible. This precludes estimation of the amount of activity transported per unit time or the velocity of transport. However, the absence of altered maximal PFK activity in the portion of nerve

TABLE 3. Sciatic nerve dry weight and contents of water, monosaccharides, and polyols in control

and diabetic rats with and without ganglioside treatment Nerve contents (nmol/mg of dry nerve)

Nerve dry weight (mg/cm)

Nerve water content (mg of H20/mg of dry nerve)

Glucose

Sorbitol

Fructose

myo-Inositol

Untreated Controls (9) Diabetics (9)

3.05 f 0.05" 2.84 f 0.05

1.68 zk 0.04 1.68 0.02

*

2.13 f 0.31 34.40 k 1.98

0.63 f 0.06 9.31 f 0.49

1.94 k 0.07 24.17 f 1.46

16.56 f 0.88' 9.85 f 0.40

Ganglioside-treated Controls (9) Diabetics (8)

3.01 f 0.19 3.00 f 0.06

1.68 zk 0.03 1.63 z i 0.06

1.84 f 0.36 38.85 f 4.74

0.46 f 0.06 9.12 f 0.50

1.43 f 0.06 23.35 f 1.48

15.55 f 0.43' 10.63 f 0.25

Data are mean f SEM values; numbers of rats are given in parentheses. Levels of significance (derived by unpaired t tests) compared with diabetic rats are as follows: 'p < 0.0 1, *p < 0.00 1. Massive differences were not tested.

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between the ligatures indicates that ligation per se did not influence PFK activity in the nerve trunk-unless local effects were restricted to the regions above the upper and below the lower ligatures. Thus, we are confident that the linear increment of PFK activity proximal to the single ligation used in the present study may be regarded as an accumulation of axonally transported enzyme activity due to interruption of that process by the nerve crush. We also believe the increase distal to the ligature to be explained by interrupted retrograde transport, but because of its smaller size and the fact that it is linear for only 24 h (Willars et al., 1987b), we concede that other factors may contribute. Deficits in proximal and distal accumulations of PFK activity in constricted sciatic nerves are seen in rats with short-term diabetes. Such deficits can be prevented by intensive insulin treatment but not by aldose reductase inhibitors (Tomlinson et al., 1987; Willars et al., 1987a). This supports further the suggestion (see above) that gangliosides and aldose reductase inhibitors influence different biochemical processes and are successful against different defects. Accumulation deficits may derive from reductions in velocity of transport, amount of material shifted, or, in the case of an enzyme, the maximal activity under the conditions of measurement. The reduction in background activity supports either of the latter two possibilities. However, the prevention of the accumulation deficit by gangliosides, without an effect on the decrease background activity. implicates reduced transport velocity as being at least partially responsible. Without more refined measurements, we can make no further suggestions about the origin of the defect in PFK activity transport. Prevention of the deficits in sciatic nerve dry weight per unit length and in background aldolase activity may indicate an effect of gangliosides giving protection against the early stages of tissue breakdown. The correlation of defective anterograde transport of proteins of the axonal endoskeleton with progressively decreasing axon diameter in streptozotocin-induced diabetic rats (Medori et al., 1985) lends powerful support to involvement of axonal transport disorders in distal axonopathy. These researchers suggested that profound retardation of transport of 30- and 60-kilodalton polypeptides may represent defective transport of glycolytic enzymes; specifically, they cite aldolase and pyruvate kinase as likely contenders. Obviously, our findings do not support a defect in aldolase transport, but the general area of transport of glycolytic enzymes merits further study. Furthermore, an investigation of the effects of ganglioside treatment on the defects revealed by the elegant methodology of Medori and her colleagues would be fruitful. Cruder experiments have shown the retardation of slow component-a of protein transport in diabetic rats to be resistant to aldose reductase inhibition (Mayer et al., 1984; Tomlinson et al., 1986). J. Neurothcm , l h l 50. No. 5 . 1988

Others have studied the effects of gangliosides on nerves of diabetic animals or on tissues subject to insults that may be a feature of the diabetic state. Thus, gangliosides are reported to protect nerve or muscle from the effects of hypoxia or ion imbalance (Vyskocil et al., 1985; Janigro et al., 1984; Bianchi et al., 1986). Disturbances of this nature are manifest in diabetic nerve (Tuck et al., 1984; Mizisin et al., 1986). Another study revealed protection by gangliosides against impaired axonal transport of different molecular forms of acetylcholinesterase in diabetic rats (Marini et al., 1986). Thus, although the neurochemical effects of gangliosides are far from clear, they appear to be of interest to those studying the aetiology of diabetic neuropathy. Acknowledgment: This study was supported jointly by Fidia Research Laboratories a n d The British Diabetic Association. W e thank Dr. Alfonsa Martelli for brisk deliveries of gangliosides and information.

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