Aldose ReductaseeCDeficient Mice Are Protected From Delayed Motor Nerve Conduction Velocity, Increased c-Jun NH2-Terminal Kinase Activation,
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糖尿病学杂志 2006年第7期
1 Institute of Molecular Biology, The University of Hong Kong, Hong Kong, Special Administrative Region (SAR), China
2 Department of Medicine, The University of Hong Kong, Hong Kong, SAR, China
3 Research Centre of Heart, Brain, Hormone and Healthy Aging, The University of Hong Kong, Hong Kong, SAR, China
4 Department of Anatomy, The University of Hong Kong, Hong Kong, SAR, China
5 Department of Pathology, Hirosaki University School of Medicine, Hirosaki, Japan
6 Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, Connecticut
7 Department of Physiology, The University of Hong Kong, Hong Kong, SAR, China
AR, aldose reductase; ARI, AR inhibitor; DHE, dihydroethidium; ERK, extracellular signaleCregulated kinase-1; JNK, c-Jun NH2 terminal kinase; MAPK, mitogen-activated protein kinase; MNCV, motor nerve conduction velocity; PAR, poly(ADP-ribose); PARP, PAR polymerase; PKC, protein kinase C; SNCV, sensory nerve conduction velocity; YFP, yellowish-green fluorescent protein
ABSTRACT
The exaggerated flux through polyol pathway during diabetes is thought to be a major cause of lesions in the peripheral nerves. Here, we used aldose reductase (AR)-deficient (AReC/eC) and AR inhibitor (ARI)-treated mice to further understand the in vivo role of polyol pathway in the pathogenesis of diabetic neuropathy. Under normal conditions, there were no obvious differences in the innervation patterns between wild-type AR (AR+/+) and AReC/eC mice. Under short-term diabetic conditions, AReC/eC mice were protected from the reduction of motor and sensory nerve conduction velocities observed in diabetic AR+/+ mice. Sorbitol levels in the sciatic nerves of diabetic AR+/+ mice were increased significantly, whereas sorbitol levels in the diabetic AReC/eC mice were significantly lower than those in diabetic AR+/+ mice. In addition, signs of oxidative stress, such as increased activation of c-Jun NH2-terminal kinase (JNK), depletion of reduced glutathione, increase of superoxide formation, and DNA damage, observed in the sciatic nerves of diabetic AR+/+ mice were not observed in the diabetic AReC/eC mice, indicating that the diabetic AReC/eC mice were protected from oxidative stress in the sciatic nerve. The diabetic AReC/eC mice also excreted less 8-hydroxy-2'-deoxyguanosine in urine than diabetic AR+/+ mice. The structural abnormalities observed in the sural nerve of diabetic AR+/+ mice were less severe in the diabetic AReC/eC mice, although it was only mildly protected by AR deficiency under short-term diabetic conditions. Signs of oxidative stress and functional and structural abnormalities were also inhibited by the ARI fidarestat in diabetic AR+/+ nerves, similar to those in diabetic AReC/eC mice. Taken together, increased polyol pathway flux through AR is a major contributing factor in the early signs of diabetic neuropathy, possibly through depletion of glutathione, increased superoxide accumulation, increased JNK activation, and DNA damage.
Neuropathy is one of the most common complications associated with diabetes (1). Although the exact mechanism for the pathogenesis of this disease is not completely understood, several contributing factors have been proposed. They include increased glucose flux through the polyol pathway, increased production of reactive oxygen species by the mitochondrial respiratory chain, nonenzymatic glycation, protein kinase C (PKC) activation, and increased flux through the hexosamine pathway (2). Among these models, the polyol pathway, which consists of aldose reductase (AR; which reduces glucose to sorbitol with the aid of NADPH) and sorbitol dehydrogenase (which converts sorbitol to fructose using the cofactor NAD+) received the most attention.
Evidence for the involvement of AR in diabetic neuropathy emerged from studies showing that AR was present in the peripheral nerves and that sorbitol was accumulated in the nerves under diabetic conditions (3). In addition, increased AR activity by introducing the human AR transgene led to more severe diabetic neuropathy (4,5) and a decreased level of reduced glutathione (5). The key evidence, however, is that several AR inhibitors (ARIs) were shown to prevent the development of diabetic neuropathy by reducing sorbitol accumulation (6), restoring blood flow (7), and improving motor nerve conduction velocity (MNCV) (6,7,8). However, the polyol pathway model still remains controversial because it has been shown that the amount of ARI that normalized the nerve sorbitol level was insufficient to normalize MNCV and nerve blood flow in diabetic rats (9).
The role of AR or the polyol pathway in diabetic neuropathy is not well understood. Previous studies demonstrated that hyperglycemia-induced oxidative stress led to the activation of mitogen-activated protein kinase (MAPK), which might contribute to the pathogenesis of this disease (10,11). Fidarestat, an ARI, was shown to prevent diabetes-induced activation of MAPK and nerve conduction velocity deficits (12), suggesting that ARIs would reduce the oxidative stress associated with diabetes. Moreover, using AR gene knockout mice (13), we have shown that AR deficiency could prevent diabetes-induced oxidative stress in nerve cells in retina (14). In addition, AR deficiency also prevented vascular abnormalities in the retina from the diabetic mice (14), suggesting that AR might contribute to diabetes-induced vascular dysfunction, which has been shown to contribute to the pathogenesis of diabetic neuropathy (15). Reduced sural endoneurial oxygen tension and diminished endoneurial blood flow have been demonstrated in diabetic animals and patients (16,17). Increased polyol pathway activity is thought to be one of the major contributors to abnormal vascular tones in diabetic animals because it could activate PKC (18,19).
To further understand the role of AR in the pathogenesis of diabetic neuropathy, we examined the effect of AR deficiency on diabetic neuropathy by making use of AReC/eC mice (13) because AR mRNA and activity are present in the mouse sciatic nerve (5,20), and sorbitol can be further accumulated in the sciatic nerve under diabetic conditions (21). The effects of an ARI, fidarestat (SNK-860; Sanwa, Nagoya, Japan), on nerve conduction velocities, signs of oxidative stress, and nerve morphologic abnormalities were also investigated in diabetic AR+/+ mice and compared with those in diabetic AReC/eC mice. Here, we found that AR deficiency and AR inhibition reduced oxidative stress in the peripheral nerves and markedly protected mice from diabetes-induced functional deficits.
RESEARCH DESIGN AND METHODS
AR+/+ and AReC/eC mice generated previously (13) and backcrossed to C57BL/6N for more than five generations were used. Animal experiments were carried out under guideline set forth by the committee on the use of live animals in teaching and research at the University of Hong Kong.
Diabetes induction.
Mice (6 weeks old, 20eC25 g) were induced to become diabetic with streptozotocin (in 0.1 mol/l citrate buffer, pH 4.5; Sigma, St. Louis, MO) by injection (200 mg/kg body wt i.p.). Control animals were injected with citrate buffer. Blood glucose was monitored with a glucose meter (Bayer, Leverkusen, Germany) 2 days later. Mice with a blood glucose level >25 mmol/l were considered diabetic, and those with blood glucose level <8 mmol/l were considered nondiabetic.
Nerve conduction velocity measurement.
The 4, 8, and 12 weeks’ diabetic AReC/eC mice and their appropriate control animals were anesthetized with Hypnorm (Janssen, Oxford, U.K.)/Hypnorval (Hoffmann-La Roche, Nutley, NJ) at a concentration of 0.1 ml per 10 g body wt (the resulting mixture of Hypnorm/Dormicum contained 1.25 mg/ml midazolam, 2.5 mg/ml fluanisone, and 0.079 mg/ml fentanyl). Body temperature was maintained automatically at a mean rectal temperature of 37.5eC37.9°C by the use of a heat pad (Fine Science Tools, Vancouver, BC, Canada). The sciatic nerve was stimulated (5eC10 V, 0.05 ms single square-wave pulses) proximally at the level of the sciatic notch and distally at the level of the ankle with platinum needle electrodes (Grass, Quincy, MA). Compound muscle action potentials were recorded from the ipsilateral foot between digits 1 and 2, amplified, stored, and displayed on a computer (Spike 2; CED, Cambridge, U.K.). The first compound action potential from individual stimulation was used for the measurement of motor latency, whereas the second one was used for the measurement of sensory latency. Averaged distal and proximal motor and sensory latencies from 10 separate recordings, together with the nerve length between the two stimulation sites, were used for calculation of MNCV and sensory nerve conduction velocity (SNCV).
Carbohydrate measurement by high-performance liquid chromatography.
The sciatic nerves of 4 weeks’ diabetic AReC/eC mice and their appropriate control animals were dissected and frozen in liquid nitrogen until processed. Dry weight of the sciatic nerves was determined after drying the nerves in a SpeedVac (AS160; Savant Instruments, Farmingdale, NY) until the weight was constant. Carbohydrates were extracted from sciatic nerves of mice as described previously (22). Carbohydrate extracts were separated on a DX500 high-performance liquid chromatography system (Dionex, Sunnyvale, CA) equipped with a Carbopac (MA-1) anion exchange column (Dionex) with isocratic elution by 420 mmol/l NaOH. Peak integration was performed, using Peaknet software (Dionex). The peak areas were normalized with the internal control, and carbohydrates were quantified against a known standard curve run in the same run. The concentrations of carbohydrates were expressed in nanomoles per milligram dry weight of sciatic nerves.
Determination of glutathione level.
Sciatic nerves were stored in liquid nitrogen until homogenization in 1 ml ice-cold 6% perchloric acid. After centrifugation at 4,000g for 10 min, nerve perchloric extracts were neutralized by 5 mol/l potassium carbonate and centrifuged at 4,000g for 5 min. The supernatant was then mixed with 0.89 ml of 20 mmol/l EDTA in 1 mol/l Tris-Cl buffer (pH 8.1). The reaction was initiated by the addition of 0.01 ml of 0-pathaldialdehyde (10 mg in 1 ml methanol; Sigma). Reaction products were detected/quantified by fluorescence spectroscopy at -excitation 345 nm and -emission 425 nm (23).
Detection of superoxide.
Dihydroethidium (DHE; 2 e蘭ol/l; Molecular Probes, Eugene, OR) was topically applied onto 6-e蘭 cryosections of tissue-freezing medium embedded sciatic nerves, incubated in a humidified chamber at 37°C (30 min, in dark). Sections were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma). Signals were captured with an Olympus IX71 microscope equipped with a Spot RT digital camera (Diagnostic Instruments, Detroit, MI).
Poly(ADP-ribose) immunostaining.
The 10-e蘭 longitudinal paraffin-embedded sections of 4% paraformaldehyde-fixed sciatic nerves from 12 weeks’ diabetic AReC/eC mice and their appropriate control mice were deparaffinized in xylene and pretreated with 0.3% H2O2 to eliminate endogenous peroxidase activity. Mouse monoclonal antieCpoly(ADP-ribose) (PAR) antibody (Alexis, San Diego, CA) was applied onto the sections at a concentration of 1:200 overnight at 4°C. PAR immunoreactivity was detected with a biotinylated anti-mouse secondary antibody and streptavidin-biotin-peroxidase complex according to the instructions from Vector Elite kit (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as substrate for peroxidase. Sections were then counterstained with hematoxylin, dehydrated, and mounted. Images were captured with an Olympus IX71 microscope equipped with a Spot RT digital camera (Diagnostic Instruments).
Protein analyses using Western blotting.
Sciatic nerves stored in liquid nitrogen were homogenized in ice-cold 0.1 mol/l NaCl, 0.05 mol/l Tris-Cl (pH 7.4), 0.001 mol/l EDTA, and a mixture of protease inhibitors (5 e蘬/ml Complete; Roche, Basel, Switzerland). SDS-PAGE (8, 10, and 15% acrylamide) was performed with 50 e蘥 of total proteins. Separated proteins were transferred to a reinforced nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) using a "tank" buffer system (Bio-Rad, Hercules, CA). Primary antibodies used were antibodies against total p46 c-Jun NH2-terminal kinase (JNK; 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), antibodies against phosphorylated p54 JNK and phosphorylated p46 JNK (1:1,000, phosphorylated on Thr-183 and Tyr-185; Cell Signaling Technology, Danvers, MA), antibodies against total extracellular signaleCregulated kinase-1 (ERK1) and ERK2 (1:1,000; Cell Signaling Technology), and antibodies against phosphorylated ERK1 and ERK2 (1:1,000, phosphorylated on Tyr-204; Santa Cruz Biotechnology). Detection was achieved using enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, U.K.). The immunoblots were scanned on a flatbed scanner (UMAX, Dallas, TX) and converted into numerical values by ImageQuant (version 5.1; Amersham Biosciences, Sunnyvale, CA).
Detection of yellowish-green fluorescent protein fluorescent nerve fibers.
AR+/+ and AReC/eC mice (13) were mated with thy1-YFP mice (24,25) to introduce yellowish-green fluorescent protein (YFP) into sensory/motor neurons and their processes for analyzing the effect of AR deficiency on the organization of peripheral nerves in live animals. The images of fluorescent YFP nerve fibers in the ear, abdomen, diaphragm, and lower leg of these mice under normal conditions were captured with a digital camera (DC500; Leica, Bensheim, Germany) that was attached to a fluorescence stereomicroscope (MZ FLIII; Leica).
Morphometric analysis of sural nerves.
Sural nerves were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide. After dehydration, midportions of the fixed sural nerves were embedded in epon and polymerized, and 1-e蘭-thick transverse nerve sections were stained with toluidine blue. Fascicular area and myelinated fiber number and size were measured at a magnification of 1,600x using a computer-assisted image analyzing system (National Institutes of Health image, Agfa Arcusscanner connected to a Quadra 700; Macintosh, Cupertino, CA).
Statistical analysis.
All data are the means ± SE. Either Student’s t test or one-way ANOVA together with Bonferroni’s post hoc test were performed. P < 0.05 was considered statistically significant.
RESULTS
The AReC/eC mice backcrossed to C57BL/6N for at least five generations had mild impairment in water reabsorption in the kidney, leading to slightly increased urine output and increased water consumption, as described previously (13). Under diabetic conditions, there was no significant difference in the urine output, water consumption, and level of blood glucose between AReC/eC and AR+/+ mice (data not shown).
AR deficiency prevented diabetes-induced MNCV and SNCV reduction.
There was no significant difference in MNCV and SNCV between nondiabetic AR+/+ and AReC/eC mice. However, after 4, 8, and 12 weeks of diabetes, AR+/+ mice showed a significant reduction of MNCV compared with their nondiabetic counterparts (P < 0.001 for each stage). However, a significant reduction of MNCV in diabetic AReC/eC mice was not observed (Fig. 1). Similarly, the reduction of SNCV observed in diabetic AR+/+ mice (P < 0.001 for each stage) was also not observed in diabetic AReC/eC mice (Fig. 1). Not surprisingly, the ARI fidarestat also protected diabetic AR+/+ mice from a reduction of MNCV almost equivalent to that of AR-deficient mutants after 12 weeks of diabetes (Fig. 1).
Effects of AR deficiency on glucose and its related metabolites in the sciatic nerve.
Nerve glucose levels in nondiabetic AR+/+ and AReC/eC mice were indistinguishable (Table 1). After 4 weeks of diabetes, both diabetic AR+/+ and AReC/eC mice showed significant increases in glucose content compared with their nondiabetic counterparts (P < 0.001). Glucose content in diabetic AR+/+ mice was not significantly different from that of diabetic AReC/eC mice (Table 1).
Under normal conditions, sorbitol and fructose contents in the sciatic nerves of AReC/eC mice were significantly reduced when compared with that of AR+/+ mice (P < 0.001). After 4 weeks of diabetes, both AR+/+ and AReC/eC mice showed significant increases in sorbitol and fructose in their sciatic nerves compared with those of the nondiabetic control animals (P < 0.001). Sorbitol and fructose levels in diabetic AReC/eC mice were significantly lower than that in diabetic AR+/+ mice (P < 0.001). The myo-inositol levels between nondiabetic AR+/+ and AReC/eC mice were similar. The myo-inositol levels in diabetic AR+/+ and AReC/eC mice were not statistically different from their nondiabetic counterparts (Table 1).
Diabetes-induced glutathione depletion in sciatic nerves was attenuated by AR deficiency.
Glutathione levels in nondiabetic AR+/+ and AReC/eC mice were similar. A significant reduction of glutathione in the 4 weeks’ diabetic AR+/+ mice was observed (P < 0.001), but no reduction in glutathione level was found in diabetic AReC/eC mice. The sciatic nerves of 12 weeks’ diabetic AReC/eC mice or ARI-treated diabetic mice also showed similar protection from the depletion of reduced glutathione in the sciatic nerves (Fig. 2). Also, we found a significant increase in the urine 8-OHdG (8-hydroxy-2'-deoxyguanosine) content in 8 weeks’ diabetic AR+/+ mice, but the increase was not statistically significant in diabetic AReC/eC mice (data not shown).
Diabetes-induced accumulation of superoxide was lower in the diabetic AReC/eC mice.
The oxidative fluorescent dye DHE, represented as red fluorescence in the nucleus of the cells, was used to determine the level of O2
· in the sciatic nerve. Under normal conditions, ethidium fluorescence was barely detectable in the nuclei of cells from AR+/+ and AReC/eC mice (6% of nuclei were positive for DHE). Sciatic nerves of 12 weeks’ diabetic AR+/+ mice showed a marked increase in DHE staining compared with that of nondiabetic AR+/+ mice. The percentage of DHE-stained nuclei was also significantly increased (P < 0.001). However, the 12 weeks’ diabetic AR+/+ mice treated with ARI or fidarestat or the diabetic AReC/eC animals showed much less accumulation of superoxide in the sciatic nerve (Fig. 3A). Quantitation results showed that the increase of DHE-positive nuclei as observed in diabetic AR+/+ mice was ameliorated in ARI-treated diabetic AR+/+ mice or diabetic AReC/eC mice (P < 0.001) (Fig. 3B).
Diabetes-induced PAR polymerase activation was less in sciatic nerves of AReC/eC mice.
To further determine other indicators of oxidative stress, PAR polymerase (PARP) reactivity, which is induced by increased free radicals and DNA damage (2), was determined by PAR immunoreactivity in nucleus. PAR-reactive nuclei were barely detectable in the sciatic nerves of nondiabetic AR+/+ and AReC/eC mice. A marked increase in PAR immunoreactivity was observed in AR+/+ mice after 12 weeks of diabetes. Moreover, 12 weeks’ diabetic AReC/eC mice were protected from PARP activation. In addition, oral treatment with the ARI fidarestat during 12 weeks of diabetes protected AR+/+ mice from PARP activation, similar to that observed in 12 weeks’ diabetic AReC/eC mice (Fig. 4).
Diabetes-induced activation of p46 JNK in the sciatic nerves was prevented by AR deficiency.
The expression level of p46 JNK in the sciatic nerve was similar in the nondiabetic AR+/+ and AReC/eC mice, and it was not affected by 4 weeks of diabetes in both groups of mice. The expression levels of phosphorylated p54 JNK and phosphorylated p46 JNK were also similar in the nondiabetic AR+/+ and AReC/eC mice. After 4 weeks of diabetes, there was a significant increase in the ratio of phosphorylated protein to total protein of p46 JNK in the diabetic AR+/+ mice when compared with the nondiabetic AR+/+ mice (P < 0.001). However, the phosphorylated proteineCtoeCtotal protein ratio of p46 JNK in the AReC/eC mice was unchanged after 4 weeks of diabetes. Unlike p46 JNK, the phosphorylated proteineCtoeCtotal protein ratio of p54 JNK was not affected after 4 weeks of diabetes in both AR+/+ and AReC/eC mice (Fig. 5). Similar analysis was also made for ERK activation, but there was no significant difference between experimental groups (data not shown).
Diabetes-induced structural abnormalities in the sural nerves were partially prevented by AR deficiency.
Under normal conditions, AR deficiency did not affect the structure of nerves, as indicated by the comparison of the total fascicular area, myelinated fiber size, and total myelinated fiber of the sural nerves of AR+/+ and AReC/eC mice (Table 2). These findings were also confirmed by analyzing the peripheral nerves of live AReC/eC mice mated with thy1-YFP transgenic mice, which have YFP protein expression in all motor and sensory neurons, including the sural nerves, and allow direct viewing of nerves in the live animals. The gross innervation patterns of major organs at all the ages studied (4, 8, 12, and 24 weeks), such as abdomen, diaphragm, kidney, bladder, ear, and lower leg, in AReC/eCmice were not obviously different from those of AR+/+ mice under normal conditions (Fig. 6), suggesting that AR deficiency does not affect the normal development of the nervous system.
In the AR+/+ mice, 8 weeks of diabetes caused a significant reduction in total fascicular area, mean myelinated fiber size, and fiber number (Table 2). Similar changes in these morphologic parameters were also observed after 12 weeks of diabetes. In the AReC/eC mice, 8 or 12 weeks of diabetes did not cause a significant reduction in fascicular area and fiber number. Mean myelinated fiber size, however, was reduced after 8 or 12 weeks of diabetes. The effect of ARI on diabetes-induced structural changes of the nerves was also tested in wild-type mice. The results showed that mice with 12 weeks of diabetes treated with ARI also showed a reduction in fascicular area and mean myelinated fiber size (Table 2). However, the reduction in fascicular area was less severe than that found in diabetic mice not treated with ARI. Furthermore, ARI treatment also protected the nerve against diabetes-induced fiber loss. However, there was no significant difference in fascicular area or total fiber number in the sural nerves from 8 and 12 weeks’ diabetic AReC/eC and AR+/+ mice or 12 weeks’ diabetic AR+/+ mice treated with ARI.
DISCUSSION
As previously described, AR-deficient mice had a mild impairment in water reabsorption in the kidney, leading to slightly increased urine output and increased water consumption under normal conditions without affecting the levels of serum electrolytes (13). However, under diabetic conditions, there was no significant difference in urine volume, water consumption, and blood and nerve glucose levels between AReC/eC and AR+/+ mice. In addition, morphometric analysis of sural nerve biopsies and gross comparisons of peripheral nerves of AR+/+ or AReC/eC mice mated with thy1-YFP transgenic mice showed no obvious effect of AR deficiency on the peripheral nervous system under normal conditions. Taken together, these observations indicate that the AReC/eC mouse model would be an invaluable model for studying the role of AR deficiency in the pathogenesis of diabetic neuropathy.
Previous studies using ARIs in experimental animal models (4,8) and diabetic patients (26) showed that inhibition of AR activity could partially restore MNCV slowing, which is one of the major features of diabetic neuropathy. Transgenic mice that overexpress AR either ubiquitously (4) or specifically in Schwann cells (5) developed more severe MNCV deficits. Here, we demonstrated that diabetic AReC/eC mice were protected against MNCV and SNCV reduction. Furthermore, ARI (fidarestat) treatment protected diabetic AR+/+ mice from MNCV reduction, similar to its protective effect on SNCV reduction, as demonstrated by others (27). Our current data suggest that exaggerated flux through the polyol pathway plays a key role in the pathogenesis of acute diabetic neuropathy, possibly acting directly on the peripheral nerves. Although the AR level in mouse sciatic nerve is low (28), the presence of AR mRNA was clearly demonstrated (5). Furthermore, in diabetic mice, AR activity (5) and sorbitol accumulation (21) were found to be increased in this tissue. The low level of sorbitol in mouse sciatic nerve does not truly reflect the level of polyol pathway activity. We have previously shown that the sorbitol level in the sciatic nerves of diabetic C57BL/10N, nondiabetic, and diabetic sorbitol dehydrogenaseeCdeficient mice were increased 4.3, 16.6, and 38.1-fold, respectively, above that of nondiabetic C57BL/10N mice (21), indicating that sorbitol is quickly converted to fructose by sorbitol dehydrogenase and that a significant amount of glucose is fluxed through the polyol pathway, particularly in the diabetic mice. Interestingly, trace amounts of sorbitol and fructose were found in the diabetic AR/sorbitol dehydrogenase double-mutant mice (data not shown) and the AReC/eC mice. These may be contributed by other proteins with AR-like activity, such as aldehyde reductase (29), the fibroblast growth factoreCregulated protein gene (Fgfrp) (30), and the androgen-regulated vas deferens protein gene (Avdp) (30).
We found that the glutathione level was decreased in the sciatic nerves of diabetic AR+/+ mice, but not in diabetic AReC/eC or ARI-treated diabetic AR+/+ mice, indicating that AR activity contributes to hyperglycemia-induced glutathione depletion. Potential mechanisms of the polyol pathway’s contribution to diabetes-induced oxidative stress have been discussed previously (2). The difference in the absolute level of glutathione reported here compared with that of the previous study (5) may be the result of differences in mouse substrains or the adaptation of a more sensitive detection method in the current study. Previous studies demonstrated that an increase of oxidative stress could lead to the activation of stress-activated protein kinases, such as MAPK (10). Our current data showed that diabetes caused a significant activation of p46 JNK in the sciatic nerves of wild-type mice, which was prevented by AR deficiency. It is likely that amelioration of oxidative stress by AR deficiency under diabetic conditions might contribute to attenuation of JNK activation in the sciatic nerve in AReC/eC mice because other signs of oxidative stress, such as superoxide accumulation (determined by DHE staining) or PARP activation (determined by PAR immunohistochemistry), in sciatic nerves observed in diabetic AR+/+ mice were also ameliorated in diabetic AReC/eC mice or ARI-treated diabetic AR+/+ mice. Taken together, our data suggest that AR is a key enzyme in the pathogenesis of diabetic neuropathy and that diabetes-induced oxidative stress might be primarily the consequence of increased flux of glucose through the polyol pathway in the nervous tissue, similar to nerve cells in the diabetic retina (14).
We observed small but significant loss of myelinated nerve fibers in our diabetic wild-type mice. Previous animal studies did not find such structural changes (4,31). This discrepancy may be due to the fact that the previous studies did not count all of the nerve fibers within the nerve fascicle. The diabetes-induced reduction of fascicular area and fiber number observed in the sural nerves of wild-type mice were largely attenuated in AReC/eC mice, indicating that AR activity contributes to these hyperglycemia-induced lesions. ARI treatment only partially alleviated these structural changes. This may be attributable to an insufficient amount of drug to completely inhibit AR in the target tissue. Interestingly, diabetes-induced reduction in mean myelinated fiber size was also not alleviated by an AR-deficiency mutation or ARI treatment, suggesting that other toxic effects of hyperglycemia, such as glycation or inappropriate activation of PKC, might contribute to this diabetes-induced structural change of the nerves. It is likely that diabetic neuropathy involves multiple pathogenic mechanisms.
Here, we demonstrate that AR or the polyol pathway plays a key role in the pathogenesis of diabetic neuropathy. Our AReC/eC mouse model is a useful model for determining its contribution to the cascade of molecular events, such as reduced number of cytoskeletons in distal axons (32eC34), transport of cytoskeltons or nerve growth factors (34), or aberrant phosphorylation (35), to further explain the role of AR deficiency in functional, biochemical, and structural recovery of peripheral nerves. Of course, these AR-deficient mice can also serve to determine the interactions of AR with other hypothesized contributors to diabetic neuropathy, such as insulinopenia, impaired neurotrophic support, or glycation.
ACKNOWLEDGMENTS
This project was partly supported by grants from the Hong Kong Research Grant Council (HKU7313/04M) and the Biotechnology Research Institute, Hong Kong University of Science and Technology (to S.K.C.).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Thomas PK: Diabetic peripheral neuropathies: their cost to patient and society and the value of knowledge of risk factors for development of interventions. Eur Neurol 41 (Suppl. 1):35eC43, 1999
Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813eC820, 2001
Gabbay KH, Merola LO, Field RA: Sorbitol pathway: presence in nerve and cord with substrate accumulation in diabetes. Science 151:209eC210, 1966
Yagihashi S, Yamagishi SI, Wada RiR, Baba M, Hohman TC, Yabe-Nishimura C, Kokai Y: Neuropathy in diabetic mice overexpressing human aldose reductase and effects of aldose reductase inhibitor. Brain 124:2448eC2458, 2001
Song Z, Fu DT, Chan YS, Leung S, Chung SS, Chung SK: Transgenic mice overexpressing aldose reductase in Schwann cells show more severe nerve conduction velocity deficit and oxidative stress under hyperglycemic stress. Mol Cell Neurosci 23:638eC647, 2003
Tomlinson DR, Moriarty RJ, Mayer JH: Prevention and reversal of defective axonal transport and motor nerve conduction velocity in rats with experimental diabetes by treatment with the aldose reductase inhibitor Sorbinil. Diabetes 33:470eC476, 1984
Nakamura J, Kato K, Hamada Y, Nakayama M, Chaya S, Nakashima E, Naruse K, Kasuya Y, Mizubayashi R, Miwa K, Yasuda Y, Kamiya H, Ienaga K, Sakakibara F, Koh N, Hotta N: A protein kinase C--selective inhibitor ameliorates neural dysfunction in streptozotocin-induced diabetic rats. Diabetes 48:2090eC2095, 1999
Yue DK, Hanwell MA, Satchell PM, Turtle JR: The effect of aldose reductase inhibition on motor nerve conduction velocity in diabetic rats. Diabetes 31:789eC794, 1982
Cameron NE, Cotter MA, Dines KC, Maxfield EK, Carey F, Mirrlees DJ: Aldose reductase inhibition, nerve perfusion, oxygenation and function in streptozotocin-diabetic rats: dose-response considerations and independence from a myo-inositol mechanism. Diabetologia 37:651eC663, 1994
Wang X, Martindale JL, Liu Y, Holbrook NJ: The cellular response to oxidative stress: influences of mitogen-activated protein kinase signalling pathways on cell survival. Biochem J 333 (Pt. 2):291eC300, 1998
Purves T, Middlemas A, Agthong S, Jude EB, Boulton AJ, Fernyhough P, Tomlinson DR: A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy. FASEB J 15:2508eC2514, 2001
Price SA, Agthong S, Middlemas AB, Tomlinson DR: Mitogen-activated protein kinase p38 mediates reduced nerve conduction velocity in experimental diabetic neuropathy: interactions with aldose reductase. Diabetes 53:1851eC1856, 2004
Ho HT, Chung SK, Law JW, Ko BC, Tam SC, Brooks HL, Knepper MA, Chung SS: Aldose reductase-deficient mice develop nephrogenic diabetes insipidus. Mol Cell Biol 20:5840eC5846, 2000
Cheung AK, Fung MK, Lam TT, So KT, Chung SS, Chung SK: Aldose reductase deficiency prevents diabetes-induced blood-retinal barrier breakdown, apoptosis, and glial reactivation in the retina of db/db mice. Diabetes 54:3119eC3125, 2005
Gooch C, Podwall D: The diabetic neuropathies. Neurologist 10:311eC322, 2004
Cameron NE, Eaton SE, Cotter MA, Tesfaye S: Vascular factors and metabolic interactions in the pathogenesis of diabetic neuropathy. Diabetologia 44:1973eC1988, 2001
Newrick PG, Wilson AJ, Jakubowski J, Boulton AJ, Ward JD: Sural nerve oxygen tension in diabetes. Br Med J (Clin Res Ed ) 293:1053eC1054, 1986
Xia P, Kramer RM, King GL: Identification of the mechanism for the inhibition of Na+,K(+)-adenosine triphosphatase by hyperglycemia involving activation of protein kinase C and cytosolic phospholipase A2. J Clin Invest 96:733eC740, 1995
Oates PJ: Polyol pathway and diabetic peripheral neuropathy. Int Rev Neurobiol 50:325eC392, 2002
Fu DTW, Lee AYW, Lin CXF, Chung SSM, Chung SS: Downregulation of mouse aldose reductase mRNA in the Schwann cells but not in the endothelial cells of sciatic nerve by hyperglycemia. Soc Neurosci Abs 22:1498, 1996
Ng TF, Lee FK, Song ZT, Calcutt NA, Lee AY, Chung SS, Chung SK, Ng DT, Lee LW: Effects of sorbitol dehydrogenase deficiency on nerve conduction in experimental diabetic mice. Diabetes 47:961eC966, 1998
Lal S, Szwergold BS, Taylor AH, Randall WC, Kappler F, Brown TR: Production of fructose and fructose-3-phosphate in maturing rat lenses. Invest Ophthalmol Vis Sci 36:969eC973, 1995
Obrosova IG, Fathallah L, Lang HJ, Greene DA: Evaluation of a sorbitol dehydrogenase inhibitor on diabetic peripheral nerve metabolism: a prevention study. Diabetologia 42:1187eC1194, 1999
Chen YS, Chung SSM, Chung SK: Noninvasive monitoring of diabetes-induced cutaneous nerve fiber loss and hypoalgesia in thy1-YFP transgenic mice. Diabetes 54:3112eC3118, 2005
Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR: Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41eC51, 2000
Judzewitsch RG, Jaspan JB, Polonsky KS, Weinberg CR, Halter JB, Halar E, Pfeifer MA, Vukadinovic C, Bernstein L, Schneider M, Liang KY, Gabbay KH, Rubenstein AH, Porte D: Aldose reductase inhibition improves nerve conduction velocity in diabetic patients. N Engl J Med 308:119eC125, 1983
Mizuno K, Kato N, Makino M, Suzuki T, Shindo M: Continuous inhibition of excessive polyol pathway flux in peripheral nerves by aldose reductase inhibitor fidarestat leads to improvement of diabetic neuropathy. J Diabetes Complications 13:141eC150, 1999
Gui T, Tanimoto T, Kokai Y, Nishimura C: Presence of a closely related subgroup in the aldo-ketoreductase family of the mouse. Eur J Biochem 227:448eC453, 1995
Sato S: Rat kidney aldose reductase and aldehyde reductase and polyol production in rat kidney. Am J Physiol 263:F799eCF805, 1992
Ho HT, Jenkins NA, Copeland NG, Gilbert DJ, Winkles JA, Louie HW, Lee FK, Chung SS, Chung SK: Comparisons of genomic structures and chromosomal locations of the mouse aldose reductase and aldose reductase-like genes. Eur J Biochem 259:726eC730, 1999
Zochodne DW, Sun H, Cheng C, Eyer J: Accelerated diabetic neuropathy in axons without neurofilaments. Brain 127:2193eC2200, 2004
Pettersen JA, Zochodne DW, Bell RB, Martin L, Hill MD: Refractory neurosarcoidosis responding to infliximab. Neurology 59:1660eC1661, 2002
Yagihashi S, Kamijo M, Watanabe K: Reduced myelinated fiber size correlates with loss of axonal neurofilaments in peripheral nerve of chronically streptozotocin diabetic rats. Am J Pathol 136:1365eC1373, 1990
Sayers NM, Beswick LJ, Middlemas A, Calcutt NA, Mizisin AP, Tomlinson DR, Fernyhough P: Neurotrophin-3 prevents the proximal accumulation of neurofilament proteins in sensory neurons of streptozocin-induced diabetic rats. Diabetes 52:2372eC2380, 2003
Fernyhough P, Gallagher A, Averill SA, Priestley JV, Hounsom L, Patel J, Tomlinson DR: Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy. Diabetes 48:881eC889, 1999(Eric C.M. Ho, Karen S.L. )
2 Department of Medicine, The University of Hong Kong, Hong Kong, SAR, China
3 Research Centre of Heart, Brain, Hormone and Healthy Aging, The University of Hong Kong, Hong Kong, SAR, China
4 Department of Anatomy, The University of Hong Kong, Hong Kong, SAR, China
5 Department of Pathology, Hirosaki University School of Medicine, Hirosaki, Japan
6 Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, Connecticut
7 Department of Physiology, The University of Hong Kong, Hong Kong, SAR, China
AR, aldose reductase; ARI, AR inhibitor; DHE, dihydroethidium; ERK, extracellular signaleCregulated kinase-1; JNK, c-Jun NH2 terminal kinase; MAPK, mitogen-activated protein kinase; MNCV, motor nerve conduction velocity; PAR, poly(ADP-ribose); PARP, PAR polymerase; PKC, protein kinase C; SNCV, sensory nerve conduction velocity; YFP, yellowish-green fluorescent protein
ABSTRACT
The exaggerated flux through polyol pathway during diabetes is thought to be a major cause of lesions in the peripheral nerves. Here, we used aldose reductase (AR)-deficient (AReC/eC) and AR inhibitor (ARI)-treated mice to further understand the in vivo role of polyol pathway in the pathogenesis of diabetic neuropathy. Under normal conditions, there were no obvious differences in the innervation patterns between wild-type AR (AR+/+) and AReC/eC mice. Under short-term diabetic conditions, AReC/eC mice were protected from the reduction of motor and sensory nerve conduction velocities observed in diabetic AR+/+ mice. Sorbitol levels in the sciatic nerves of diabetic AR+/+ mice were increased significantly, whereas sorbitol levels in the diabetic AReC/eC mice were significantly lower than those in diabetic AR+/+ mice. In addition, signs of oxidative stress, such as increased activation of c-Jun NH2-terminal kinase (JNK), depletion of reduced glutathione, increase of superoxide formation, and DNA damage, observed in the sciatic nerves of diabetic AR+/+ mice were not observed in the diabetic AReC/eC mice, indicating that the diabetic AReC/eC mice were protected from oxidative stress in the sciatic nerve. The diabetic AReC/eC mice also excreted less 8-hydroxy-2'-deoxyguanosine in urine than diabetic AR+/+ mice. The structural abnormalities observed in the sural nerve of diabetic AR+/+ mice were less severe in the diabetic AReC/eC mice, although it was only mildly protected by AR deficiency under short-term diabetic conditions. Signs of oxidative stress and functional and structural abnormalities were also inhibited by the ARI fidarestat in diabetic AR+/+ nerves, similar to those in diabetic AReC/eC mice. Taken together, increased polyol pathway flux through AR is a major contributing factor in the early signs of diabetic neuropathy, possibly through depletion of glutathione, increased superoxide accumulation, increased JNK activation, and DNA damage.
Neuropathy is one of the most common complications associated with diabetes (1). Although the exact mechanism for the pathogenesis of this disease is not completely understood, several contributing factors have been proposed. They include increased glucose flux through the polyol pathway, increased production of reactive oxygen species by the mitochondrial respiratory chain, nonenzymatic glycation, protein kinase C (PKC) activation, and increased flux through the hexosamine pathway (2). Among these models, the polyol pathway, which consists of aldose reductase (AR; which reduces glucose to sorbitol with the aid of NADPH) and sorbitol dehydrogenase (which converts sorbitol to fructose using the cofactor NAD+) received the most attention.
Evidence for the involvement of AR in diabetic neuropathy emerged from studies showing that AR was present in the peripheral nerves and that sorbitol was accumulated in the nerves under diabetic conditions (3). In addition, increased AR activity by introducing the human AR transgene led to more severe diabetic neuropathy (4,5) and a decreased level of reduced glutathione (5). The key evidence, however, is that several AR inhibitors (ARIs) were shown to prevent the development of diabetic neuropathy by reducing sorbitol accumulation (6), restoring blood flow (7), and improving motor nerve conduction velocity (MNCV) (6,7,8). However, the polyol pathway model still remains controversial because it has been shown that the amount of ARI that normalized the nerve sorbitol level was insufficient to normalize MNCV and nerve blood flow in diabetic rats (9).
The role of AR or the polyol pathway in diabetic neuropathy is not well understood. Previous studies demonstrated that hyperglycemia-induced oxidative stress led to the activation of mitogen-activated protein kinase (MAPK), which might contribute to the pathogenesis of this disease (10,11). Fidarestat, an ARI, was shown to prevent diabetes-induced activation of MAPK and nerve conduction velocity deficits (12), suggesting that ARIs would reduce the oxidative stress associated with diabetes. Moreover, using AR gene knockout mice (13), we have shown that AR deficiency could prevent diabetes-induced oxidative stress in nerve cells in retina (14). In addition, AR deficiency also prevented vascular abnormalities in the retina from the diabetic mice (14), suggesting that AR might contribute to diabetes-induced vascular dysfunction, which has been shown to contribute to the pathogenesis of diabetic neuropathy (15). Reduced sural endoneurial oxygen tension and diminished endoneurial blood flow have been demonstrated in diabetic animals and patients (16,17). Increased polyol pathway activity is thought to be one of the major contributors to abnormal vascular tones in diabetic animals because it could activate PKC (18,19).
To further understand the role of AR in the pathogenesis of diabetic neuropathy, we examined the effect of AR deficiency on diabetic neuropathy by making use of AReC/eC mice (13) because AR mRNA and activity are present in the mouse sciatic nerve (5,20), and sorbitol can be further accumulated in the sciatic nerve under diabetic conditions (21). The effects of an ARI, fidarestat (SNK-860; Sanwa, Nagoya, Japan), on nerve conduction velocities, signs of oxidative stress, and nerve morphologic abnormalities were also investigated in diabetic AR+/+ mice and compared with those in diabetic AReC/eC mice. Here, we found that AR deficiency and AR inhibition reduced oxidative stress in the peripheral nerves and markedly protected mice from diabetes-induced functional deficits.
RESEARCH DESIGN AND METHODS
AR+/+ and AReC/eC mice generated previously (13) and backcrossed to C57BL/6N for more than five generations were used. Animal experiments were carried out under guideline set forth by the committee on the use of live animals in teaching and research at the University of Hong Kong.
Diabetes induction.
Mice (6 weeks old, 20eC25 g) were induced to become diabetic with streptozotocin (in 0.1 mol/l citrate buffer, pH 4.5; Sigma, St. Louis, MO) by injection (200 mg/kg body wt i.p.). Control animals were injected with citrate buffer. Blood glucose was monitored with a glucose meter (Bayer, Leverkusen, Germany) 2 days later. Mice with a blood glucose level >25 mmol/l were considered diabetic, and those with blood glucose level <8 mmol/l were considered nondiabetic.
Nerve conduction velocity measurement.
The 4, 8, and 12 weeks’ diabetic AReC/eC mice and their appropriate control animals were anesthetized with Hypnorm (Janssen, Oxford, U.K.)/Hypnorval (Hoffmann-La Roche, Nutley, NJ) at a concentration of 0.1 ml per 10 g body wt (the resulting mixture of Hypnorm/Dormicum contained 1.25 mg/ml midazolam, 2.5 mg/ml fluanisone, and 0.079 mg/ml fentanyl). Body temperature was maintained automatically at a mean rectal temperature of 37.5eC37.9°C by the use of a heat pad (Fine Science Tools, Vancouver, BC, Canada). The sciatic nerve was stimulated (5eC10 V, 0.05 ms single square-wave pulses) proximally at the level of the sciatic notch and distally at the level of the ankle with platinum needle electrodes (Grass, Quincy, MA). Compound muscle action potentials were recorded from the ipsilateral foot between digits 1 and 2, amplified, stored, and displayed on a computer (Spike 2; CED, Cambridge, U.K.). The first compound action potential from individual stimulation was used for the measurement of motor latency, whereas the second one was used for the measurement of sensory latency. Averaged distal and proximal motor and sensory latencies from 10 separate recordings, together with the nerve length between the two stimulation sites, were used for calculation of MNCV and sensory nerve conduction velocity (SNCV).
Carbohydrate measurement by high-performance liquid chromatography.
The sciatic nerves of 4 weeks’ diabetic AReC/eC mice and their appropriate control animals were dissected and frozen in liquid nitrogen until processed. Dry weight of the sciatic nerves was determined after drying the nerves in a SpeedVac (AS160; Savant Instruments, Farmingdale, NY) until the weight was constant. Carbohydrates were extracted from sciatic nerves of mice as described previously (22). Carbohydrate extracts were separated on a DX500 high-performance liquid chromatography system (Dionex, Sunnyvale, CA) equipped with a Carbopac (MA-1) anion exchange column (Dionex) with isocratic elution by 420 mmol/l NaOH. Peak integration was performed, using Peaknet software (Dionex). The peak areas were normalized with the internal control, and carbohydrates were quantified against a known standard curve run in the same run. The concentrations of carbohydrates were expressed in nanomoles per milligram dry weight of sciatic nerves.
Determination of glutathione level.
Sciatic nerves were stored in liquid nitrogen until homogenization in 1 ml ice-cold 6% perchloric acid. After centrifugation at 4,000g for 10 min, nerve perchloric extracts were neutralized by 5 mol/l potassium carbonate and centrifuged at 4,000g for 5 min. The supernatant was then mixed with 0.89 ml of 20 mmol/l EDTA in 1 mol/l Tris-Cl buffer (pH 8.1). The reaction was initiated by the addition of 0.01 ml of 0-pathaldialdehyde (10 mg in 1 ml methanol; Sigma). Reaction products were detected/quantified by fluorescence spectroscopy at -excitation 345 nm and -emission 425 nm (23).
Detection of superoxide.
Dihydroethidium (DHE; 2 e蘭ol/l; Molecular Probes, Eugene, OR) was topically applied onto 6-e蘭 cryosections of tissue-freezing medium embedded sciatic nerves, incubated in a humidified chamber at 37°C (30 min, in dark). Sections were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma). Signals were captured with an Olympus IX71 microscope equipped with a Spot RT digital camera (Diagnostic Instruments, Detroit, MI).
Poly(ADP-ribose) immunostaining.
The 10-e蘭 longitudinal paraffin-embedded sections of 4% paraformaldehyde-fixed sciatic nerves from 12 weeks’ diabetic AReC/eC mice and their appropriate control mice were deparaffinized in xylene and pretreated with 0.3% H2O2 to eliminate endogenous peroxidase activity. Mouse monoclonal antieCpoly(ADP-ribose) (PAR) antibody (Alexis, San Diego, CA) was applied onto the sections at a concentration of 1:200 overnight at 4°C. PAR immunoreactivity was detected with a biotinylated anti-mouse secondary antibody and streptavidin-biotin-peroxidase complex according to the instructions from Vector Elite kit (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as substrate for peroxidase. Sections were then counterstained with hematoxylin, dehydrated, and mounted. Images were captured with an Olympus IX71 microscope equipped with a Spot RT digital camera (Diagnostic Instruments).
Protein analyses using Western blotting.
Sciatic nerves stored in liquid nitrogen were homogenized in ice-cold 0.1 mol/l NaCl, 0.05 mol/l Tris-Cl (pH 7.4), 0.001 mol/l EDTA, and a mixture of protease inhibitors (5 e蘬/ml Complete; Roche, Basel, Switzerland). SDS-PAGE (8, 10, and 15% acrylamide) was performed with 50 e蘥 of total proteins. Separated proteins were transferred to a reinforced nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) using a "tank" buffer system (Bio-Rad, Hercules, CA). Primary antibodies used were antibodies against total p46 c-Jun NH2-terminal kinase (JNK; 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), antibodies against phosphorylated p54 JNK and phosphorylated p46 JNK (1:1,000, phosphorylated on Thr-183 and Tyr-185; Cell Signaling Technology, Danvers, MA), antibodies against total extracellular signaleCregulated kinase-1 (ERK1) and ERK2 (1:1,000; Cell Signaling Technology), and antibodies against phosphorylated ERK1 and ERK2 (1:1,000, phosphorylated on Tyr-204; Santa Cruz Biotechnology). Detection was achieved using enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, U.K.). The immunoblots were scanned on a flatbed scanner (UMAX, Dallas, TX) and converted into numerical values by ImageQuant (version 5.1; Amersham Biosciences, Sunnyvale, CA).
Detection of yellowish-green fluorescent protein fluorescent nerve fibers.
AR+/+ and AReC/eC mice (13) were mated with thy1-YFP mice (24,25) to introduce yellowish-green fluorescent protein (YFP) into sensory/motor neurons and their processes for analyzing the effect of AR deficiency on the organization of peripheral nerves in live animals. The images of fluorescent YFP nerve fibers in the ear, abdomen, diaphragm, and lower leg of these mice under normal conditions were captured with a digital camera (DC500; Leica, Bensheim, Germany) that was attached to a fluorescence stereomicroscope (MZ FLIII; Leica).
Morphometric analysis of sural nerves.
Sural nerves were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide. After dehydration, midportions of the fixed sural nerves were embedded in epon and polymerized, and 1-e蘭-thick transverse nerve sections were stained with toluidine blue. Fascicular area and myelinated fiber number and size were measured at a magnification of 1,600x using a computer-assisted image analyzing system (National Institutes of Health image, Agfa Arcusscanner connected to a Quadra 700; Macintosh, Cupertino, CA).
Statistical analysis.
All data are the means ± SE. Either Student’s t test or one-way ANOVA together with Bonferroni’s post hoc test were performed. P < 0.05 was considered statistically significant.
RESULTS
The AReC/eC mice backcrossed to C57BL/6N for at least five generations had mild impairment in water reabsorption in the kidney, leading to slightly increased urine output and increased water consumption, as described previously (13). Under diabetic conditions, there was no significant difference in the urine output, water consumption, and level of blood glucose between AReC/eC and AR+/+ mice (data not shown).
AR deficiency prevented diabetes-induced MNCV and SNCV reduction.
There was no significant difference in MNCV and SNCV between nondiabetic AR+/+ and AReC/eC mice. However, after 4, 8, and 12 weeks of diabetes, AR+/+ mice showed a significant reduction of MNCV compared with their nondiabetic counterparts (P < 0.001 for each stage). However, a significant reduction of MNCV in diabetic AReC/eC mice was not observed (Fig. 1). Similarly, the reduction of SNCV observed in diabetic AR+/+ mice (P < 0.001 for each stage) was also not observed in diabetic AReC/eC mice (Fig. 1). Not surprisingly, the ARI fidarestat also protected diabetic AR+/+ mice from a reduction of MNCV almost equivalent to that of AR-deficient mutants after 12 weeks of diabetes (Fig. 1).
Effects of AR deficiency on glucose and its related metabolites in the sciatic nerve.
Nerve glucose levels in nondiabetic AR+/+ and AReC/eC mice were indistinguishable (Table 1). After 4 weeks of diabetes, both diabetic AR+/+ and AReC/eC mice showed significant increases in glucose content compared with their nondiabetic counterparts (P < 0.001). Glucose content in diabetic AR+/+ mice was not significantly different from that of diabetic AReC/eC mice (Table 1).
Under normal conditions, sorbitol and fructose contents in the sciatic nerves of AReC/eC mice were significantly reduced when compared with that of AR+/+ mice (P < 0.001). After 4 weeks of diabetes, both AR+/+ and AReC/eC mice showed significant increases in sorbitol and fructose in their sciatic nerves compared with those of the nondiabetic control animals (P < 0.001). Sorbitol and fructose levels in diabetic AReC/eC mice were significantly lower than that in diabetic AR+/+ mice (P < 0.001). The myo-inositol levels between nondiabetic AR+/+ and AReC/eC mice were similar. The myo-inositol levels in diabetic AR+/+ and AReC/eC mice were not statistically different from their nondiabetic counterparts (Table 1).
Diabetes-induced glutathione depletion in sciatic nerves was attenuated by AR deficiency.
Glutathione levels in nondiabetic AR+/+ and AReC/eC mice were similar. A significant reduction of glutathione in the 4 weeks’ diabetic AR+/+ mice was observed (P < 0.001), but no reduction in glutathione level was found in diabetic AReC/eC mice. The sciatic nerves of 12 weeks’ diabetic AReC/eC mice or ARI-treated diabetic mice also showed similar protection from the depletion of reduced glutathione in the sciatic nerves (Fig. 2). Also, we found a significant increase in the urine 8-OHdG (8-hydroxy-2'-deoxyguanosine) content in 8 weeks’ diabetic AR+/+ mice, but the increase was not statistically significant in diabetic AReC/eC mice (data not shown).
Diabetes-induced accumulation of superoxide was lower in the diabetic AReC/eC mice.
The oxidative fluorescent dye DHE, represented as red fluorescence in the nucleus of the cells, was used to determine the level of O2
· in the sciatic nerve. Under normal conditions, ethidium fluorescence was barely detectable in the nuclei of cells from AR+/+ and AReC/eC mice (6% of nuclei were positive for DHE). Sciatic nerves of 12 weeks’ diabetic AR+/+ mice showed a marked increase in DHE staining compared with that of nondiabetic AR+/+ mice. The percentage of DHE-stained nuclei was also significantly increased (P < 0.001). However, the 12 weeks’ diabetic AR+/+ mice treated with ARI or fidarestat or the diabetic AReC/eC animals showed much less accumulation of superoxide in the sciatic nerve (Fig. 3A). Quantitation results showed that the increase of DHE-positive nuclei as observed in diabetic AR+/+ mice was ameliorated in ARI-treated diabetic AR+/+ mice or diabetic AReC/eC mice (P < 0.001) (Fig. 3B).
Diabetes-induced PAR polymerase activation was less in sciatic nerves of AReC/eC mice.
To further determine other indicators of oxidative stress, PAR polymerase (PARP) reactivity, which is induced by increased free radicals and DNA damage (2), was determined by PAR immunoreactivity in nucleus. PAR-reactive nuclei were barely detectable in the sciatic nerves of nondiabetic AR+/+ and AReC/eC mice. A marked increase in PAR immunoreactivity was observed in AR+/+ mice after 12 weeks of diabetes. Moreover, 12 weeks’ diabetic AReC/eC mice were protected from PARP activation. In addition, oral treatment with the ARI fidarestat during 12 weeks of diabetes protected AR+/+ mice from PARP activation, similar to that observed in 12 weeks’ diabetic AReC/eC mice (Fig. 4).
Diabetes-induced activation of p46 JNK in the sciatic nerves was prevented by AR deficiency.
The expression level of p46 JNK in the sciatic nerve was similar in the nondiabetic AR+/+ and AReC/eC mice, and it was not affected by 4 weeks of diabetes in both groups of mice. The expression levels of phosphorylated p54 JNK and phosphorylated p46 JNK were also similar in the nondiabetic AR+/+ and AReC/eC mice. After 4 weeks of diabetes, there was a significant increase in the ratio of phosphorylated protein to total protein of p46 JNK in the diabetic AR+/+ mice when compared with the nondiabetic AR+/+ mice (P < 0.001). However, the phosphorylated proteineCtoeCtotal protein ratio of p46 JNK in the AReC/eC mice was unchanged after 4 weeks of diabetes. Unlike p46 JNK, the phosphorylated proteineCtoeCtotal protein ratio of p54 JNK was not affected after 4 weeks of diabetes in both AR+/+ and AReC/eC mice (Fig. 5). Similar analysis was also made for ERK activation, but there was no significant difference between experimental groups (data not shown).
Diabetes-induced structural abnormalities in the sural nerves were partially prevented by AR deficiency.
Under normal conditions, AR deficiency did not affect the structure of nerves, as indicated by the comparison of the total fascicular area, myelinated fiber size, and total myelinated fiber of the sural nerves of AR+/+ and AReC/eC mice (Table 2). These findings were also confirmed by analyzing the peripheral nerves of live AReC/eC mice mated with thy1-YFP transgenic mice, which have YFP protein expression in all motor and sensory neurons, including the sural nerves, and allow direct viewing of nerves in the live animals. The gross innervation patterns of major organs at all the ages studied (4, 8, 12, and 24 weeks), such as abdomen, diaphragm, kidney, bladder, ear, and lower leg, in AReC/eCmice were not obviously different from those of AR+/+ mice under normal conditions (Fig. 6), suggesting that AR deficiency does not affect the normal development of the nervous system.
In the AR+/+ mice, 8 weeks of diabetes caused a significant reduction in total fascicular area, mean myelinated fiber size, and fiber number (Table 2). Similar changes in these morphologic parameters were also observed after 12 weeks of diabetes. In the AReC/eC mice, 8 or 12 weeks of diabetes did not cause a significant reduction in fascicular area and fiber number. Mean myelinated fiber size, however, was reduced after 8 or 12 weeks of diabetes. The effect of ARI on diabetes-induced structural changes of the nerves was also tested in wild-type mice. The results showed that mice with 12 weeks of diabetes treated with ARI also showed a reduction in fascicular area and mean myelinated fiber size (Table 2). However, the reduction in fascicular area was less severe than that found in diabetic mice not treated with ARI. Furthermore, ARI treatment also protected the nerve against diabetes-induced fiber loss. However, there was no significant difference in fascicular area or total fiber number in the sural nerves from 8 and 12 weeks’ diabetic AReC/eC and AR+/+ mice or 12 weeks’ diabetic AR+/+ mice treated with ARI.
DISCUSSION
As previously described, AR-deficient mice had a mild impairment in water reabsorption in the kidney, leading to slightly increased urine output and increased water consumption under normal conditions without affecting the levels of serum electrolytes (13). However, under diabetic conditions, there was no significant difference in urine volume, water consumption, and blood and nerve glucose levels between AReC/eC and AR+/+ mice. In addition, morphometric analysis of sural nerve biopsies and gross comparisons of peripheral nerves of AR+/+ or AReC/eC mice mated with thy1-YFP transgenic mice showed no obvious effect of AR deficiency on the peripheral nervous system under normal conditions. Taken together, these observations indicate that the AReC/eC mouse model would be an invaluable model for studying the role of AR deficiency in the pathogenesis of diabetic neuropathy.
Previous studies using ARIs in experimental animal models (4,8) and diabetic patients (26) showed that inhibition of AR activity could partially restore MNCV slowing, which is one of the major features of diabetic neuropathy. Transgenic mice that overexpress AR either ubiquitously (4) or specifically in Schwann cells (5) developed more severe MNCV deficits. Here, we demonstrated that diabetic AReC/eC mice were protected against MNCV and SNCV reduction. Furthermore, ARI (fidarestat) treatment protected diabetic AR+/+ mice from MNCV reduction, similar to its protective effect on SNCV reduction, as demonstrated by others (27). Our current data suggest that exaggerated flux through the polyol pathway plays a key role in the pathogenesis of acute diabetic neuropathy, possibly acting directly on the peripheral nerves. Although the AR level in mouse sciatic nerve is low (28), the presence of AR mRNA was clearly demonstrated (5). Furthermore, in diabetic mice, AR activity (5) and sorbitol accumulation (21) were found to be increased in this tissue. The low level of sorbitol in mouse sciatic nerve does not truly reflect the level of polyol pathway activity. We have previously shown that the sorbitol level in the sciatic nerves of diabetic C57BL/10N, nondiabetic, and diabetic sorbitol dehydrogenaseeCdeficient mice were increased 4.3, 16.6, and 38.1-fold, respectively, above that of nondiabetic C57BL/10N mice (21), indicating that sorbitol is quickly converted to fructose by sorbitol dehydrogenase and that a significant amount of glucose is fluxed through the polyol pathway, particularly in the diabetic mice. Interestingly, trace amounts of sorbitol and fructose were found in the diabetic AR/sorbitol dehydrogenase double-mutant mice (data not shown) and the AReC/eC mice. These may be contributed by other proteins with AR-like activity, such as aldehyde reductase (29), the fibroblast growth factoreCregulated protein gene (Fgfrp) (30), and the androgen-regulated vas deferens protein gene (Avdp) (30).
We found that the glutathione level was decreased in the sciatic nerves of diabetic AR+/+ mice, but not in diabetic AReC/eC or ARI-treated diabetic AR+/+ mice, indicating that AR activity contributes to hyperglycemia-induced glutathione depletion. Potential mechanisms of the polyol pathway’s contribution to diabetes-induced oxidative stress have been discussed previously (2). The difference in the absolute level of glutathione reported here compared with that of the previous study (5) may be the result of differences in mouse substrains or the adaptation of a more sensitive detection method in the current study. Previous studies demonstrated that an increase of oxidative stress could lead to the activation of stress-activated protein kinases, such as MAPK (10). Our current data showed that diabetes caused a significant activation of p46 JNK in the sciatic nerves of wild-type mice, which was prevented by AR deficiency. It is likely that amelioration of oxidative stress by AR deficiency under diabetic conditions might contribute to attenuation of JNK activation in the sciatic nerve in AReC/eC mice because other signs of oxidative stress, such as superoxide accumulation (determined by DHE staining) or PARP activation (determined by PAR immunohistochemistry), in sciatic nerves observed in diabetic AR+/+ mice were also ameliorated in diabetic AReC/eC mice or ARI-treated diabetic AR+/+ mice. Taken together, our data suggest that AR is a key enzyme in the pathogenesis of diabetic neuropathy and that diabetes-induced oxidative stress might be primarily the consequence of increased flux of glucose through the polyol pathway in the nervous tissue, similar to nerve cells in the diabetic retina (14).
We observed small but significant loss of myelinated nerve fibers in our diabetic wild-type mice. Previous animal studies did not find such structural changes (4,31). This discrepancy may be due to the fact that the previous studies did not count all of the nerve fibers within the nerve fascicle. The diabetes-induced reduction of fascicular area and fiber number observed in the sural nerves of wild-type mice were largely attenuated in AReC/eC mice, indicating that AR activity contributes to these hyperglycemia-induced lesions. ARI treatment only partially alleviated these structural changes. This may be attributable to an insufficient amount of drug to completely inhibit AR in the target tissue. Interestingly, diabetes-induced reduction in mean myelinated fiber size was also not alleviated by an AR-deficiency mutation or ARI treatment, suggesting that other toxic effects of hyperglycemia, such as glycation or inappropriate activation of PKC, might contribute to this diabetes-induced structural change of the nerves. It is likely that diabetic neuropathy involves multiple pathogenic mechanisms.
Here, we demonstrate that AR or the polyol pathway plays a key role in the pathogenesis of diabetic neuropathy. Our AReC/eC mouse model is a useful model for determining its contribution to the cascade of molecular events, such as reduced number of cytoskeletons in distal axons (32eC34), transport of cytoskeltons or nerve growth factors (34), or aberrant phosphorylation (35), to further explain the role of AR deficiency in functional, biochemical, and structural recovery of peripheral nerves. Of course, these AR-deficient mice can also serve to determine the interactions of AR with other hypothesized contributors to diabetic neuropathy, such as insulinopenia, impaired neurotrophic support, or glycation.
ACKNOWLEDGMENTS
This project was partly supported by grants from the Hong Kong Research Grant Council (HKU7313/04M) and the Biotechnology Research Institute, Hong Kong University of Science and Technology (to S.K.C.).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Thomas PK: Diabetic peripheral neuropathies: their cost to patient and society and the value of knowledge of risk factors for development of interventions. Eur Neurol 41 (Suppl. 1):35eC43, 1999
Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813eC820, 2001
Gabbay KH, Merola LO, Field RA: Sorbitol pathway: presence in nerve and cord with substrate accumulation in diabetes. Science 151:209eC210, 1966
Yagihashi S, Yamagishi SI, Wada RiR, Baba M, Hohman TC, Yabe-Nishimura C, Kokai Y: Neuropathy in diabetic mice overexpressing human aldose reductase and effects of aldose reductase inhibitor. Brain 124:2448eC2458, 2001
Song Z, Fu DT, Chan YS, Leung S, Chung SS, Chung SK: Transgenic mice overexpressing aldose reductase in Schwann cells show more severe nerve conduction velocity deficit and oxidative stress under hyperglycemic stress. Mol Cell Neurosci 23:638eC647, 2003
Tomlinson DR, Moriarty RJ, Mayer JH: Prevention and reversal of defective axonal transport and motor nerve conduction velocity in rats with experimental diabetes by treatment with the aldose reductase inhibitor Sorbinil. Diabetes 33:470eC476, 1984
Nakamura J, Kato K, Hamada Y, Nakayama M, Chaya S, Nakashima E, Naruse K, Kasuya Y, Mizubayashi R, Miwa K, Yasuda Y, Kamiya H, Ienaga K, Sakakibara F, Koh N, Hotta N: A protein kinase C--selective inhibitor ameliorates neural dysfunction in streptozotocin-induced diabetic rats. Diabetes 48:2090eC2095, 1999
Yue DK, Hanwell MA, Satchell PM, Turtle JR: The effect of aldose reductase inhibition on motor nerve conduction velocity in diabetic rats. Diabetes 31:789eC794, 1982
Cameron NE, Cotter MA, Dines KC, Maxfield EK, Carey F, Mirrlees DJ: Aldose reductase inhibition, nerve perfusion, oxygenation and function in streptozotocin-diabetic rats: dose-response considerations and independence from a myo-inositol mechanism. Diabetologia 37:651eC663, 1994
Wang X, Martindale JL, Liu Y, Holbrook NJ: The cellular response to oxidative stress: influences of mitogen-activated protein kinase signalling pathways on cell survival. Biochem J 333 (Pt. 2):291eC300, 1998
Purves T, Middlemas A, Agthong S, Jude EB, Boulton AJ, Fernyhough P, Tomlinson DR: A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy. FASEB J 15:2508eC2514, 2001
Price SA, Agthong S, Middlemas AB, Tomlinson DR: Mitogen-activated protein kinase p38 mediates reduced nerve conduction velocity in experimental diabetic neuropathy: interactions with aldose reductase. Diabetes 53:1851eC1856, 2004
Ho HT, Chung SK, Law JW, Ko BC, Tam SC, Brooks HL, Knepper MA, Chung SS: Aldose reductase-deficient mice develop nephrogenic diabetes insipidus. Mol Cell Biol 20:5840eC5846, 2000
Cheung AK, Fung MK, Lam TT, So KT, Chung SS, Chung SK: Aldose reductase deficiency prevents diabetes-induced blood-retinal barrier breakdown, apoptosis, and glial reactivation in the retina of db/db mice. Diabetes 54:3119eC3125, 2005
Gooch C, Podwall D: The diabetic neuropathies. Neurologist 10:311eC322, 2004
Cameron NE, Eaton SE, Cotter MA, Tesfaye S: Vascular factors and metabolic interactions in the pathogenesis of diabetic neuropathy. Diabetologia 44:1973eC1988, 2001
Newrick PG, Wilson AJ, Jakubowski J, Boulton AJ, Ward JD: Sural nerve oxygen tension in diabetes. Br Med J (Clin Res Ed ) 293:1053eC1054, 1986
Xia P, Kramer RM, King GL: Identification of the mechanism for the inhibition of Na+,K(+)-adenosine triphosphatase by hyperglycemia involving activation of protein kinase C and cytosolic phospholipase A2. J Clin Invest 96:733eC740, 1995
Oates PJ: Polyol pathway and diabetic peripheral neuropathy. Int Rev Neurobiol 50:325eC392, 2002
Fu DTW, Lee AYW, Lin CXF, Chung SSM, Chung SS: Downregulation of mouse aldose reductase mRNA in the Schwann cells but not in the endothelial cells of sciatic nerve by hyperglycemia. Soc Neurosci Abs 22:1498, 1996
Ng TF, Lee FK, Song ZT, Calcutt NA, Lee AY, Chung SS, Chung SK, Ng DT, Lee LW: Effects of sorbitol dehydrogenase deficiency on nerve conduction in experimental diabetic mice. Diabetes 47:961eC966, 1998
Lal S, Szwergold BS, Taylor AH, Randall WC, Kappler F, Brown TR: Production of fructose and fructose-3-phosphate in maturing rat lenses. Invest Ophthalmol Vis Sci 36:969eC973, 1995
Obrosova IG, Fathallah L, Lang HJ, Greene DA: Evaluation of a sorbitol dehydrogenase inhibitor on diabetic peripheral nerve metabolism: a prevention study. Diabetologia 42:1187eC1194, 1999
Chen YS, Chung SSM, Chung SK: Noninvasive monitoring of diabetes-induced cutaneous nerve fiber loss and hypoalgesia in thy1-YFP transgenic mice. Diabetes 54:3112eC3118, 2005
Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR: Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41eC51, 2000
Judzewitsch RG, Jaspan JB, Polonsky KS, Weinberg CR, Halter JB, Halar E, Pfeifer MA, Vukadinovic C, Bernstein L, Schneider M, Liang KY, Gabbay KH, Rubenstein AH, Porte D: Aldose reductase inhibition improves nerve conduction velocity in diabetic patients. N Engl J Med 308:119eC125, 1983
Mizuno K, Kato N, Makino M, Suzuki T, Shindo M: Continuous inhibition of excessive polyol pathway flux in peripheral nerves by aldose reductase inhibitor fidarestat leads to improvement of diabetic neuropathy. J Diabetes Complications 13:141eC150, 1999
Gui T, Tanimoto T, Kokai Y, Nishimura C: Presence of a closely related subgroup in the aldo-ketoreductase family of the mouse. Eur J Biochem 227:448eC453, 1995
Sato S: Rat kidney aldose reductase and aldehyde reductase and polyol production in rat kidney. Am J Physiol 263:F799eCF805, 1992
Ho HT, Jenkins NA, Copeland NG, Gilbert DJ, Winkles JA, Louie HW, Lee FK, Chung SS, Chung SK: Comparisons of genomic structures and chromosomal locations of the mouse aldose reductase and aldose reductase-like genes. Eur J Biochem 259:726eC730, 1999
Zochodne DW, Sun H, Cheng C, Eyer J: Accelerated diabetic neuropathy in axons without neurofilaments. Brain 127:2193eC2200, 2004
Pettersen JA, Zochodne DW, Bell RB, Martin L, Hill MD: Refractory neurosarcoidosis responding to infliximab. Neurology 59:1660eC1661, 2002
Yagihashi S, Kamijo M, Watanabe K: Reduced myelinated fiber size correlates with loss of axonal neurofilaments in peripheral nerve of chronically streptozotocin diabetic rats. Am J Pathol 136:1365eC1373, 1990
Sayers NM, Beswick LJ, Middlemas A, Calcutt NA, Mizisin AP, Tomlinson DR, Fernyhough P: Neurotrophin-3 prevents the proximal accumulation of neurofilament proteins in sensory neurons of streptozocin-induced diabetic rats. Diabetes 52:2372eC2380, 2003
Fernyhough P, Gallagher A, Averill SA, Priestley JV, Hounsom L, Patel J, Tomlinson DR: Aberrant neurofilament phosphorylation in sensory neurons of rats with diabetic neuropathy. Diabetes 48:881eC889, 1999(Eric C.M. Ho, Karen S.L. )