Polyol Pathway Activity Research Articles

The Renal Association. Bristol, England. March 26–27, 1998

Thickening and reduplication of the tubular basement membrane has been reported as an early event in diabetic nephropathy. In the current study we have examined the polar requirements of proximal tubular cells for the D-glucose stimulated accumulation of fibronectin. We also examined the mechanism by which glucose led to accumulation of fibronectin, with particular emphasis on the polyol pathway. Incubation of confluent monolayers of LLC-PK1 cells grown on tissue culture inserts with 25 mM D-glucose on either their apical or basolateral aspect, led to fibronectin accumulation in the basolateral compartment. This reached statistical significance 24 h following apical addition of glucose (2.7 fold increase compared to 5 mM D-glucose, p = 0.007, n = 6), and 12 h after the basolateral addition of glucose (2.54 fold increase compared to 5 mM D-glucose, p = 0.02, n = 6). The increase in fibronectin concentration in response to glucose was inhibited by the aldose reductase inhibitor sorbinil. At a dose of 100&mgr;M sorbinil there was 59% inhibition of fibronectin accumulation in response to glucose, 48 h after the addition of the inhibitor (4.76 +/- 1.4 vs 11.53 +/- 1.41, mean +/- SD, p = 0.01, n = 3). Exposure of cells to glucose at either their apical or basolateral aspect lead to accumulation of intracellular glucose and polyol pathway activation, as assessed by sorbitol accumulation. Accumulation of intracellular glucose and hence subsequent polyol pathway activation occurred independently of transport of glucose by either apical sodium linked glucose transporter (SLGT) or basolateral GLUT 1. The data demonstrate that fibronectin generation in response to glucose was non-polar in terms application of glucose, but polar in terms of fibronectin accumulation. Furthermore modulation of fibronectin was mediated by polyol pathway activation, and more specifically related to the metabolism of sorbitol to fructose.

Molecular mechanisms of glucose action on angiotensinogen gene expression in rat proximal tubular cells

Clinical studies have shown that the angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists decrease proteinuria and slow the progression of nephropathy in diabetes, indicating that Ang II plays an important role in the development of nephropathy. We have previously reported that high levels of glucose stimulate the expression of rat angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells. We hypothesized that the stimulatory effect of D(+)-glucose on the expression of the ANG gene in kidney proximal tubular cells is mediated via de novo synthesis of diacylglycerol (DAG) and the protein kinase C (PKC) signal transduction pathway. Immortalized rat proximal tubular cells (IRPTCs) were cultured in monolayer. The stimulatory effect of glucose on the activation of polyol pathway and PKC signal transduction pathway in IRPTCs was determined. The immunoreactive rat ANG (IR-rANG) in the culture medium and the cellular ANG mRNA were measured with a specific radioimmunoassay and a reverse transcription-polymerase chain reaction assay, respectively. D(+)-glucose (25 mM) markedly increased the intracellular levels of sorbitol, fructose, DAG, and PKC activity as well as the expression of IR-rANG and ANG mRNA in IRPTCs. These stimulatory effects of D(+)-glucose (25 mM) were blocked by an inhibitor of aldose reductase, Tolrestat. PKC inhibitors also inhibited the stimulatory effect of D(+)-glucose (25 mM) on the expression of the IR-rANG in IRPTCs. The addition of phorbol 12-myristate 13-acetate further enhanced the stimulatory effect of D(+)-glucose (25 mM) on the expression of the IR-rANG in IRPTCs and blocked the inhibitory effect of Tolrestat. These studies suggest that the stimulatory effect of a high level of D(+)-glucose (25 mM) on the expression of the ANG gene in IRPTCs is mediated, at least in part, via the de novo synthesis of DAG, an activator of PKC signal transduction pathway.

Renal proximal tubular cell fibronectin accumulation in response to glucose is polyol pathway dependent

Thickening and reduplication of the tubular basement membrane have been reported as early events in diabetic nephropathy. In this study, we have examined the polar requirements of proximal tubular cells for the d-glucose-stimulated accumulation of fibronectin and the mechanism by which this occurred, with particular emphasis on the polyol pathway. To determine the polarity of fibronectin generation in response to glucose, LLC-PK1 cells were grown on porous tissue culture inserts. Monolayer confluence was determined by serial measurement of transepithelial resistance. Confluent cells were growth arrested by serum deprivation, and all experiments were performed under serum-free conditions. Application of 25 mm d-glucose to either the apical or basolateral aspect of LLC-PK1 cells led to fibronectin accumulation in the basolateral compartment. This reached statistical significance 24 hours following apical addition of glucose (2.6-fold increase compared with 5 mm d-glucose, P = 0.0025, N = 6 vs. N = 4 controls) and 12 hours after the basolateral addition of glucose (2.5-fold increase compared with 5 mm d-glucose, P = 0.03, N = 6 vs. N = 4 controls). Exposure of cells to glucose at either their apical or basolateral aspect leads to accumulation of intracellular glucose and polyol pathway activation, as assessed by sorbitol accumulation. The increase in fibronectin concentration in response to glucose was inhibited by the aldose reductase inhibitor sorbinil. At a dose of 100 micron sorbinil, there was a 59% inhibition of fibronectin accumulation in response to apical glucose (P = 0.004, N = 3 sorbinil vs. N = 4 controls) and a 66% inhibition in response to basolateral glucose (P = 0.008, N = 3 sorbinil vs. N = 4 controls) 48 hours after the addition of the inhibitor. Furthermore, fibronectin accumulation was also demonstrated following both the apical and basolateral addition of 1 mm sorbitol, but not following the addition of 25 mm galactose to either aspect of the cells. Following the addition of sorbitol, there was a 2. 8-fold increase in fibronectin 48 hours after apical stimulation (P = 0.01, N = 3 treated vs. N = 4 control) and a 2.27-fold increase following basolateral stimulation (P = 0.04, N = 3 treated vs. N = 4 control) at 24 hours. In summary, these data demonstrate that fibronectin generation in response to glucose was nonpolar in terms of application of glucose but was polar in terms of fibronectin accumulation. The mechanisms of glucose-induced modulation of fibronectin were mediated by polyol pathway activation and were more specifically related to the metabolism of sorbitol to fructose.

STUDY OF RENAL SORBITOLDEHYDROGENASE IN EXPERIMENTAL DIABETIC NEPHROPATHY

The link between the polyol pathway and the ocular complications of diabetes mellitus is explained by the excessive storage of sorbitol and the release of osmotic stress. The renal complications could also be explained by the osmotic hypothesis, but the polyol pathway activity is reduced in this case. The study of sorbitol dehydrogenase (SDH) activity, one of the enzymes involved in the catabolism of glucose by this pathway in renal and hepatic homogenates from diabetic animals, shows a constant increase of the hepatic enzyme activity compared to that at the renal level. The different variations of the renal SDH activity can be explained by the effect of hyperglycemia on the active form of the enzyme and its inactivation by nonenzymatic glycosylation.

Stimulation of nerve growth factor and substance P expression in the iris–trigeminal axis of diabetic rats—involvement of oxidative stress and effects of aldose reductase inhibition

In rats with streptozotocin-induced diabetes, we measured increased (by 61%; P<0.05) mRNA for nerve growth factor (NGF) in the iris together with increased (by 82%; P<0.05) mRNA for preprotachykinin (the substance P precursor) in the trigeminal ganglion, suggesting that increased NGF was driving increased substance P gene expression. In other diabetic rats, these changes were prevented by treatment with either an antioxidant (butylated hydroxytoluene; 1% by diet) or an aldose reductase inhibitor (ARI) (sorbinil; 25 mg/kg/day p.o.) and the sorbinil treatment was associated with significant inhibition of polyol pathway intermediates in both lens and sciatic nerve. This suggests that polyol pathway activity in the lens may translate to oxidative stress-driving stimulation of NGF gene expression in the iris. The change is selective for NGF, because expression of the analogous neurotrophin, neurotrophin-3 (NT-3), was unaltered in the same irises. These changes suggest that oxidative stress and/or inflammation can drive up NGF expression in diabetes—a mechanism that might participate in iritis.

Effect of the Aldose Reductase Inhibitor Tolrestat on Nerve Conduction Velocity, NA/K ATPase Activity, and Polyols in Red Blood Cells, Sciatic Nerve, Kidney Cortex, and Kidney Medulla of Diabetic Rats

Long-term prospective studies comparing the effects of conventional and intensive insulin therapy have linked diabetic hyperglycemia to the development of diabetic retinopathy, nephropathy, and neuropathy. The mechanisms through which glucose metabolism leads to the development of these secondary complications, however, are incompletely understood. In animal models of diabetic neuropathy, the loss of nerve function in myelinated nerve fibers has been related to a series of biochemical changes. Nerve glucose, which is in equilibrium with plasma glucose levels, rapidly increases during diabetic hyperglycemia because glucose entry is independent of insulin. This excess glucose is metabolized in large part by the polyol pathway. Increased flux through this pathway is accompanied by the depletion of myo-inositol, a loss of Na/K ATPase activity and the accumulation of sodium. Supportive evidence linking these biochemical changes to the loss of nerve function has come from studies in which aldose reductase inhibitors block polyol pathway activity, prevent the depletion of myo-inositol and the accumulation of sodium and preserve Na/K ATPase activity, as well as nerve function. The kidney and red blood cells (RBCs) are two additional sites of diabetic lesions that have been reported to develop biochemical changes similar to those in the nerve. We observed that polyol levels in the kidney cortex, medulla, and RBCs increased two- to ninefold in rats following 10 weeks of untreated diabetes. Polyol accumulation was accompanied by a 30% decrease in myo-inositol levels in the kidney cortex, but no change in RBCs or the kidney medulla. Na/K ATPase activity was decreased by 59% in RBCs but was unaffected in the kidney cortex or medulla. Aldose reductase inhibitor treatment that preserved myo-inositol levels, Na/K ATPase, and conduction velocity in the sciatic nerve also preserved Na/K ATPase activity in RBCs. Our results suggest that the pathophysiologic mechanisms underlying diabetic neuropathy are different from those of diabetic nephropathy. Our results also suggest that RBCs maybe a surrogate tissue for the assessment of diabetes-induced changes in nerve Na/K ATPase activity.

Electroretinogram in sucrose-fed diabetic rats treated with an aldose reductase inhibitor or an anticoagulant.

To investigate the role of increased polyol pathway activity and hemodynamic deficits in the pathogenesis of diabetic retinopathy in non-insulin-dependent diabetes mellitus (NIDDM), Otsuka Long-Evans Tokushima fatty (OLETF) rats, an animal model of human NIDDM, were given water with or without 30% sucrose and some of them were fed laboratory chow containing 0.03% cilostazol, an anticoagulant, or 0.05% [5-(3-thienyl)tetrazol-1-yl] acetic acid monohydrate (TAT), an aldose reductase inhibitor, for 8 wk. Long-Evans Tokushima Otsuka (LETO) rats were used as nondiabetic controls. The peak latencies of oscillatory potentials of the electroretinogram in sucrose-fed OLETF rats were significantly prolonged compared with those in OLETF rats without sucrose feeding and LETO rats. There was a marked increase in platelet aggregability and a significant decrease in erythrocyte 2,3-diphosphoglycerate in sucrose-fed OLETF rats. Cilostazol significantly improved these parameters without changes in retinal levels of sorbitol and fructose. TAT, however, ameliorated all of these parameters. These findings confirm that the sucrose-fed OLETF rat is a useful animal model of retinopathy in human NIDDM and suggest that cilostazol improved diabetic retinopathy by modifying vascular factors, not by altering polyol pathway activity.

Polyol pathway activation and glutathione redox status in non—insulin-dependent diabetic patients

The current study aimed to evaluate whether nicotinamide adenine dinucleotide phosphate (NADPH) alteration in erythrocytes from patients with non—insulin-dependent diabetes mellitus (NIDDM) is responsible for the impaired glutathione (GSH) redox status, and to assess if short-term inhibition of the polyol pathway normalizes NADPH levels and GSH redox status via an amelioration of the NADPH/total NADP (tNADP) ratio. For this purpose, erythrocyte NADPH and GSH levels were measured in 18 NIDDM patients at baseline and then after 1 week of random double-blind assignment to treatment with either tolrestat (an aldose reductase inhibitor, 200 mg daily) (n = 12) or placebo (n = 6). A group of 16 healthy volunteers served as the control. In the basal condition, mean GSH ( P < .0001) and NADPH ( P < .0001) levels and NADPH/tNADP ( P < .0001) and GSH/glutathione disulfide (GSSG) ( P < .005) ratios were lower in NIDDM patients than in control subjects. Tolrestat treatment increased GSH levels ( P < .05 v placebo and baseline) and the NADPH/tNADP ratio ( P < .05 v placebo and baseline). Interestingly, tolrestat-induced changes in GSH and NADPH levels and in GSH/GSSG and NADPH/tNADP ratios were significant only in patients who showed a decreased NADPH/tNADP ratio at baseline (n = 8). In these latter patients, we also found a direct correlation between percentage increments in GSH levels and NADPH/tNADP ratios after tolrestat treatment ( r = .71, P < .05). In conclusion, our findings support the hypothesis that polyol pathway activation decreases NADPH and GSH levels. Accordingly, short-term inhibition of this enzymatic route increased both the GSH level and the NADPH/tNADP ratio. These changes were observable only in the subgroup of patients with an abnormal NADPH/tNADP ratio at baseline. Polyol pathway inhibition could be useful for decreasing oxidative stress in NIDDM.

Oxidative stress and antioxidant treatment in diabetic neuropathy

Oxidative stress is one of the factors contributing to the development of diabetic neuropathy. Several mechanisms, among which glucose autooxidation, glycation of antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase, and increased polyol pathway activity, lead to increased production of reactive oxygen species. Both direct toxicity of oxygen free radicals to the peripheral nerve as well as changes in endothelial function and vascular reactivity, possibly by the quenching of nitric oxide, may lead to nerve dysfunction. Antioxidant drugs like iron chelators, N-acetylcysteine, probucol, α-lipoic acid and vitamin E can prevent nerve dysfunction in experimental diabetes. The present review focuses on the potential mechanisms explaining the association between oxidative stress and diabetic neuropathy, and summarizes the studies in which the effects of antioxidant treatment on the diabetic peripheral nerve have been evaluated. © 1997 John Wiley & Sons, Ltd.

Schwann cell changes induced as early as one week after galactose intoxication

In galactose neuropathy, aldose reductase inhibitor (ARI)-preventable Schwann cell injury has been reported in studies in which galactose feeding continued over a period of months. Given the link between these morphologic changes and polyol pathway flux, polyol accumulation after just days of galactose feeding points to the possibility that structural changes occur much earlier than previously reported. The aim of this study was to examine rat sciatic nerve after 7 days of galactose feeding for evidence of myelinated fiber injury and establish whether it is related to polyol accumulation. Compared to control or ARI-treated galactose-fed rats, nerves from untreated galactose-fed rats had increased water (P < 0.05) ad dulcitol (P < 0.008) content and decreased amounts of myo-inositol (P < 0.01). Electron microscopy revealed reactive Schwann cell changes in myelinated fibers characterized by increased cytoplasmic volume, and the occurrence of lipid droplets pi granules of Reich and enlarged mitochondria. Dystrophic accumulation of intermediate filaments was also observed in the inner glial loop. Degenerative changes included periaxonal swelling, enlarged mitochondria without recognizable cristae, lysis of Schwann cell cytoplasm and demyelination. Reactive (P < 0.05) and degenerative (P < 0.01) changes as well as the number of redundant basal lamina profiles (P < 0.05) were significantly more frequent in untreated galactose-fed rats compared to controls. ARI treatment attenuated these changes. Consistent with the initial stages of onion-bulb formation, profiles with imbricate Schwann cells were also seen only in untreated galactose-fed rats. The findings suggest that short-term increases in polyol pathway activity can have deleterious effects Schwann cells of myelinated fibers.

Effect of tolrestat, an aldose reductase inhibitor, on neutrophil respiratory burst activity in diabetic patients

One hypothesis for the reduction in oxidative killing of neutrophils in diabetic patients is that increased polyol pathway activity during hyperglycemia reduces intracellular levels of nicotinamide adenine dinucleotide phosphate (NADPH), resulting in the reduction of neutrophil superoxide production during the respiratory burst. To test this hypothesis, we assessed the effect of tolrestat, an aldose reductase inhibitor, on neutrophil respiratory burst activity (NRBA) in diabetic patients. We measured fasting plasma glucose (FPG), hemoglobin A 1 (HbA 1), and NRBA levels in 79 diabetic patients and 48 normal controls. NRBA was reassessed in 34 patients after 4 weeks of tolrestat or placebo treatment, in seven controls after 4 weeks of tolrestat treatment, and in seven patients after 4 weeks of blood glucose control. NRBA was determined by flow cytometry, which detected fluorescent 2′,7′-dichlorofluorescein (DCF) in neutrophils formed from 2′,7′-dichlorofluorescein diacetate (DCF-DA) during phorbol myristate acetate (PMA)-induced respiratory bursts. Diabetic patients showed lower NRBA than the normal controls (mean cellular fluorescence, 438 ± 103 v 668 ± 101, mean ± SD, P < .001). NRBA, in diabetic patients showed a negative correlation with HbA 1 ( r = −.336, P < .005). Tolrestat treatment for 4 weeks in 17 patients restored the reduced NRBA to an almost normal level (relative NRBA, 0.55 ± 0.20 v 0.99 ± 0.36, P < .05) despite the fact that FPG level did not change (11.8 ± 2.8 v 11.4 ± 2.8 mmol/L). NRBA of these patients after tolrestat treatment was not significantly different from that of seven control subjects treated with tolrestat for 4 weeks. In 17 placebo-treated patients, there were no significant changes in NRBA and FPG level. The vigorous blood glucose control for 4 weeks in seven patients (16.6 ± 2.1 v 8.6 ± 2.3 mmol/L) also restored the reduced NRBA to almost normal (relative NRBA, 0.55 ± 0.21 v 0.90 ± 0.30, P < .05). The result that the reduced NRBA in diabetic patients was restored to almost normal either by tolrestat treatment or by blood glucose control strongly supports the hypothesis of this study.

Effect of inhibition of aldose reductase on glucose flux, diacylglycerol formation, protein kinase C, and phospholipase A2 activation

Activation of the polyol pathway under hyperglycemic conditions is proposed to contribute to the development of diabetic nephropathy. The mechanisms by which this activation may lead to functional and structural changes within the kidney are yet to be definitively established. We have examined in vitro the steps linking increased polyol pathway activity resulting from hyperglycemia to prostaglandin production. Following the demonstration of increased prostaglandin E (PGE) levels in glomeruli from diabetic rats (14.9 ± 2.5 v 59.1 ± 19.4 ng PGE/mg protein), a specific inhibitor of aldose reductase, HOE-843, was used in vitro to analyze the response to hyperglycemia of the steps preceding prostaglandin production. In explants of glomeruli from control animals, increasing the glucose concentration in vitro from 5.6 mmol/L to 25 mmol/L resulted in a significant increase in the flux of glucose through the pentose phosphate pathway ([PPP] 1.29 ± 0.08 v 2.00 ± 0.11 nmol/h), de novo diacylglycerol synthesis (2.2 ± 0.1 v 3.1 ± 0.2 μmol/mg protein), membrane protein kinase C (PKC) activity (18.7 ± 0.5 v 24.3 ± 0.75 pmol/μg protein), and in vitro phospholipase A 2 (PLA 2) activity (2.18 ± 0.46 v 3.83 ± 1.07 nmol arachidonic acid hydrolyzed/min/mg cytosolic protein). For all parameters measured, the increase resulting from the increased glucose concentration could be prevented by in vitro addition of HOE-843 for 24 hours before measurement. These findings provide evidence to suggest a mechanism linking increased polyol pathway activity and an increase in PLA 2 activity to increased prostaglandin production, which is observed in diabetes of recent onset and may ultimately lead to changes associated with the development of diabetic nephropathy.

The aldose reductase inhibitor sorbinil does not prevent the impairment in nitric oxide-mediated neurotransmission in anococcygeus muscle from diabetic rats.

This study investigated whether increased polyol pathway activity could contribute to alterations in nitrergic neurotransmission in anococcygeus muscles from 8-week diabetic rats. In the presence of guanethidine (10-30 microM) and clonidine (0.01-0.05 microM), relaxations obtained to nitrergic nerve stimulation (0.5-5 Hz, 10-s train), to sodium nitroprusside (5-500 nM) and to nitric oxide (0.1-3 microM) were significantly reduced in muscles from diabetic rats compared to responses from control rats. Treatment of diabetic rats with the aldose reductase inhibitor sorbinil (42 mg/kg per day via feed for 8 weeks) did not affect impaired reactivity to nitrergic nerve stimulation, sodium nitroprusside or nitric oxide. The results suggest increased polyol pathway activity does not contribute to the alterations in nitrergic neurotransmission in anococcygeus muscles from diabetic rats.

Effect of hyperglycemia on the polyol pathway in rat kidney during the perinatal period.

The activity of the polyol pathway was studied in developing rat kidney. For this purpose, sorbitol content, aldose-reductase activity and sorbitol-dehydrogenase activity were determined in papilla from fetuses and 24-h-old neonates. After birth, no significant difference was observed in sorbitol content, whereas sorbitol-dehydrogenase activity decreased and aldose-reductase activity doubled. Changes in aldose-reductase activity were due to an increased number of enzymatic sites but not with a change in affinity. Low levels of sorbitol were found in fetal and neonatal medulla together with low levels of urine osmolarity. In neonates, sorbitol contents were tenfold lower than in the adult, probably as a result of a lower affinity and a lower number of enzymatic aldose-reductase sites. Attempts to increase the activity of polyol pathway in fetal kidney were made by means of hyperglycemic animals; this approach resulted in an increase of aldose-reductase activity without any change in sorbitol content. Our results indicate that, in fetal and neonatal kidneys, aldose-reductase activity is probably not the limiting factor for sorbitol synthesis; another parameter, such as the availability of NADPH, might explain the low efficiency of the polyol pathway during the perinatal period.

Glycation, oxidative stress, and scavenger activity: glucose metabolism and radical scavenger dysfunction in endothelial cells.

It has been reported that oxidative stress is increased in vivo in the diabetic state. Increased oxidative stress is caused not only by accelerated production of oxygen-free radicals but also by decreased scavenging of those molecules. Endothelial cells are extremely sensitive to oxidative stress, resulting in impairments of various endothelial cell function. In this report, we studied the association of intracellular glucose metabolism and oxygen radical scavenging function via the glutathione redox (GR) cycle in cells exposed to high-glucose conditions using cultured human umbilical vein endothelial cells. Glutathione-dependent H2O2 degradation in cells exposed to 33 mmol/l glucose (HG) for 5-7 days was reduced by 48% vs. 5.5 mmol/l glucose (NG). This impairment under the oxidative stress was D-glucose-specific and concentration-dependent and was also associated with a 42% decrease in intracellular NADPH content. Exposure of cells to 200 micromol/l H2O2 stimulated the GR cycle and the pentose phosphate pathway (PPP) at the same time. In the HG condition, activation of PPP was reduced by 50%, which was consistent with a decrease in NADPH content. Inhibition of glycolysis by H2O2 was less marked in HG cells versus NG cells. Activation of polyol pathway in HG cells is not responsible for the decrease in intracellular NADPH content. These results indicate that activation of the PPP and NADPH supply to the GR cycle is impaired in HG cells exposed to H2O2, which may result in increased oxidative stress to endothelial cells.

Influence of aldose reductase inhibition on the microvascular reactivity in experimental diabetes

1. 1. To verify if tolrestat, an aldose reductase inhibitor, corrects the impaired responses of microvessels to histamine and bradykinin in alloxan-diabetic rats, the mesenteric microcirculation was studied in vivo in anaesthetised animals. 2. 2. The impaired responses were corrected by tolrestat 5 mg/kg/day for 7 days p.o. Similar responses to acetylcholine and sodium nitroprusside were obtained in preparations of diabetic and control rats and were not altered by tolrestat treatment. 3. 3. As in diabetes, galactosemia induced impaired responses to histamine and bradykinin; these altered responses were corrected by tolrestat treatment. 4. 4. These data allow us to suggest that the polyol pathway activity might be involved in the altered responses of microvessels observed in diabetic rats. It is possible that polyol activation may play an important role in the development of vascular dysfunction in diabetes mellitus.

The effects of acetyl- l-carnitine and sorbinil on peripheral nerve structure, chemistry, and function in experimental diabetes

Nerve conduction velocity (NCV) increased with age in nondiabetic male Wistar rats for the first 26 weeks of life. The NCV of animals made hyperglycemic at age 6 weeks by administration of streptozotocin (STZ) also increases, but at a slower rate. Animals with 4 weeks of hyperglycemia and reduced NCV treated with an aldose reductase inhibitor (sorbinil) or a short-chain acyl-carnitine (acetyl- l-carnitine [ALC]) daily for 16 weeks showed an improvement in NCV. Morphometric studies of tibial nerves collected from animals after 20 weeks of hyperglycemia (age 26 weeks) showed a consistent reduction in the width of the myelin sheath and little change in axon area. The number of large myelinated fibers (>6.5 μm) found in nerves collected from hyperglycemic animals was less than the number found in nondiabetic animals. Treatment of hyperglycemic rats with either sorbinil or ALC was associated with increased NCV, myelin width, and large myelinated fibers. The apparent metabolic effect of these agents was similar for fatty acid metabolism, but different for polyol pathway activity. We conclude that in animals hyperglycemic long enough to slow NCV, sorbinil and/or ALC treatment reduces the functional, structural, and biochemical changes associated with hyperglycemia that occur in the myelin sheath.

Galactosemic Neuropathy in Transgenic Mice for Human Aldose Reductase

We studied the functional consequences of an enhanced polyol pathway activity, elicited with galactose feeding, on the peripheral nerve of transgenic mice expressing human aldose reductase. Nontransgenic littermate mice were used as controls. With a quantitative immunoassay, the expression level of human aldose reductase in the sciatic nerve was 791 +/- 44 ng/mg protein (mean +/- SE), about 25% of that in human sural nerve. When the transgenic mice were fed food containing 30% galactose, significant levels of galactitol accumulated in the sciatic nerve. Galactose feeding of nontransgenic littermate mice led to a 10-fold lower accumulation of galactitol. Galactose feeding for 16 weeks caused a significant and progressive decrease in motor nerve conduction velocity in transgenic mice to 80% of the level of galactose-fed littermate mice, which was not significantly different from that of galactose-free littermate mice. A morphometric analysis of sciatic nerve detected > 10% reduction of mean myelinated fiber size but no alterations of myelinated fiber density in galactose-fed transgenic mice compared with other groups. The functional and structural changes that develop in galactose-fed transgenic mice are similar to those previously reported in diabetic animals. The results of these studies suggest that transgenic mice expressing human aldose reductase may be a useful model not only for defining the role of the polyol pathway in diabetic neuropathy but also for identifying and characterizing effective inhibitors specific for human aldose reductase.

Impaired myelinated fiber regeneration following freeze-injury in rats with streptozotocin-induced diabetes: involvement of the polyol pathway

This study examined the effects of streptozotocin-induced diabetes mellitus and aldose reductase inhibitor (ZD5522) treatment on myelinated fiber regeneration in rats. After 2 months of diabetes, with or without ZD5522 treatment (10 mg · kg −1 · day −1) from induction, sciatic nerve degeneration was initiated by a punctate lesion using a liquid NZ 2 cooled probe. Regeneration was studied over a subsequent 14-day period using in vitro electrophysiological techniques. There was a 21.4% ( P < 0.001) deficit in the maximum fiber regeneration distance in diabetic rats, 14 days postlesion. This was partially (64.9%, P < 0.01) prevented by aldose reductase inhibitor treatment, the resultant regeneration distance being not significantly different from that of age-matched nondiabetic control rats. The regeneration rate, assessed from data collected 4, 9 and 14 days postlesion, was 23.7% ( P < 0.001) reduced by diabetes and ZD5522 treatment provided 73.1% protection ( P < 0.01). We conclude that polyol pathway activity is involved in impaired regeneration in experimental diabetes, potential pathophysiological mechanisms involving a reduction in neurotrophic support and impaired endoneurial blood supply.

Nerve function in galactosaemic rats: effects of evening primrose oil and doxazosin

Rats were fed for 6 weeks with a 40% galactose diet to chronically stimulate the polyol pathway. Sciatic motor and saphenous sensory nerve conduction velocity deficits of 22% and 14% respectively developed. Treatment with evening primrose oil or doxazosin from galactosaemia induction partially (approximately 60%) prevented the development of reduced motor and sensory conduction, the former treatment being more successful than the latter. Sciatic nerve resistance to hypoxic conduction failure was 49% increased by galactosaemia. This abnormality was 27% and 43% prevented by doxazosin and evening primrose oil respectively. Galactosaemic sciatic nerves had a 10% increase in water content and endoneurial capillary density was 24% reduced. While neither treatment affected water content, both caused angiogenesis, elevating capillary density by approximately 16%. The data support the hypothesis that, as in experimental diabetes mellitus, the main effect of polyol pathway activation on peripheral nerve function occurs indirectly via a neurovascular action.