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The generation of Reactive Oxygen Species (ROS) and Advanced Glycation Endproducts (AGE’s) resulting from chronic tissue exposure to elevated levels of glucose has been identified as a key modulator of diabetic complications including diabetic cardiomyopathy. Abnormal activation of the polyol pathway converts excess glucose to sorbitol, generating ROS and AGEs. This conversion is catalyzed by the enzyme Aldose Reductase. Inhibition of Aldose Reductase, the rate limiting enzyme in the polyol pathway, reduces the production of sorbitol, attenuates increases in NADH, and down-regulates the synthesis of ROS. Methods and Results: Cultured human adult cells (NHK) were exposed to elevated glucose [25mM] to simulate hyperglycemic conditions in diabetic tissue in the presence/absence of Aldose Reductase inhibitor AT-001 [0.18nM]. AT-001 dose selection was based on previous dose response studies. Cytosolic oxidative stress was evaluated and quantified using dihydroethidium (DHE) staining and quantitated via colorimetric assessment. Mitochondrial-specific oxidative stress ROS levels were quantitated using MitoSOX staining. AT-001 prevented the production and accumulation of ROS as assessed by both DHE quantitation and MitoSOX staining, demonstrating effective inhibition of polyol pathway associated damage to these cells. Conclusions: AT-001 effectively prevented cellular damage caused by oxidative stress under hyperglycemic conditions warranting further investigation of AT-001 in the treatment of diabetic complications. AT-001 is currently being evaluated in a pivotal phase 2/3 study (ARISE-HF NCT 04083339) to treat diabetic cardiomyopathy and prevent progression to overt heart failure and also includes sub-analyses for retinopathy and diabetic peripheral neuropathy. Disclosure R. Perfetti: Stock/Shareholder; Self; Applied Therapeutics. G. Yepuri: None. N.A. Quadri: None. R. Ramasamy: Consultant; Self; Applied Therapeutics. A.F. Ghannam: Employee; Self; Applied Therapeutics Inc. Stock/Shareholder; Self; Sanofi US. S. Shendelman: Employee; Self; Applied Therapeutics. Stock/Shareholder; Self; Applied Therapeutics. Funding Applied Therapeutics (NCT04083339)

It is demonstrated that glucose fluctuations, such as postprandial hyperglycemia, and hypoglycemia due to diabetes treatment could be implicated in the development of diabetic complications. There is evidence for the involvement of oxidative stress in the pathogenesis of diabetic neuropathy. It has been elucidated that hyperglycemia induced oxidative stress and caused peripheral nerve dysfunction. However, the effects of hypoglycemia and blood glucose fluctuation on diabetic neuropathy remain unclear. In the present study, we investigated the mechanisms of recurrent short-term hypoglycemia and hyperglycemia on apoptosis and oxidative stress in Schwann cells, and we also examined the involvement of polyol pathway and endoplasmic reticulum (ER) stress pathway in these abnormalities. Immortalized adult mouse Schwann cells were exposed to five different glucose treatments over 3 days: 1) normal glucose (NG), 2) constant low glucose (LG), 3) constant high glucose (HG), 4) intermittent low glucose (ILG; 1 h three times per day), 5) intermittent high glucose (IHG; 1 h three times per day). Cell viability was reduced not only HG, but also LG. Furthermore, either IHG or ILG also decreased cell viabilities. TBARS levels were increased by HG, LG, IHG, and ILG. High glucose (HG and IHG) and low glucose (LG and ILG) increased the expression of cleaved caspase-3 and reduced that of Bcl-2. Epalrestat, an aldose reductase inhibitor, as well as 4-PBA, an ER stress inhibitor, recovered cell death and oxidative stress which were induced by LG, HG, ILG and IHG. These results suggest that intermittent hypoglycemia and hyperglycemia enhanced apoptosis and oxidative stress through both polyol pathway and ER stress pathway in Schwann cells, and that not only hyperglycemia, but also hypoglycemia and glucose fluctuations might cause nerve dysfunction in diabetes, resulting in the onset and progression of diabetic neuropathy. Disclosure A. Kato: None. Y. Tatsumi: None. H. Yako: None. T. Himeno: None. M. Kondo: None. Y. Kato: Speaker's Bureau; Self; Merck & Co., Inc. H. Kamiya: Speaker's Bureau; Self; Astellas Pharma Inc., Eli Lilly Japan K.K., MSD K.K., Novartis Pharma K.K., Novo Nordisk Oharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K. K. Sango: None. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceu. Co. Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. K. Kato: None. Funding Japan Society for the Promotion of Science

Hyperactivation of the polyol pathway (PP) contributes to development of diabetic complications. Aldose reductase (AR) is the rate-controlling enzyme in PP that catalyzes NADPH-dependent reduction of glucose to sorbitol. In hyperglycemic and ischemic conditions, PP activation causes intracellular sorbitol accumulation leading to osmotic stress, cell death and diabetic complications. We developed AT-001 based on further characterization of AR and structural changes within the active site of AR following enzymatic activation to design molecular AR inhibitors with enhanced specificity, affinity and selectivity compared with previous AR inhibitors that failed to inhibit redox-activated AR and were associated with off-target effects and/or lack of efficacy. AT-001 safety, pharmacokinetics (PK) and efficacy were assessed in a phase 1/2 study comprised of single oral ascending doses (SAD, n=8 per group) of 5, 10, 20 and 40 mg/kg once daily (QD) and multiple doses (MAD) of placebo (Pbo) or AT-001 5, 20, 40 mg/kg QD or 20 mg/kg twice daily (BID) for 7 d. Subjects with type 2 diabetes, age 18-75 and HbA1c 5.0-8.5% were allocated to each dose cohort (n=8 AT-001, n=2 Pbo). AT-001 was well-tolerated with no drug-related AEs or changes in liver or renal function up to the highest dose tested. Mean half-life (T1/2) and time of maximum concentration (Tmax) ranged from 1.7-3h and 1.75-3h, respectively in both SAD and day 1 MAD with no accumulation at day 7. AR inhibition was confirmed by significant dose-dependent reductions in whole blood sorbitol in SAD and MAD up to approximately -50% change from baseline in SAD and days 1 and 7 of MAD vs. a -3% change in Pbo (p Disclosure S. Shendelman: Stock/Shareholder; Self; Applied Therapeutics Inc. Other Relationship; Self; Applied Therapeutics Inc. R. Perfetti: Employee; Self; Applied Therapeutics Inc., Sanofi. Stock/Shareholder; Self; Applied Therapeutics Inc., Sanofi.

OBJECTIVE Impairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome. RESEARCH DESIGN AND METHODS Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS). RESULTS Proteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a complex I protein) were reduced by 29 and 36% (P < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control. CONCLUSIONS Abnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.

OBJECTIVE Prevention of diabetic retinopathy would benefit from availability of drugs that preempt the effects of hyperglycemia on retinal vessels. We aimed to identify candidate drug targets by investigating the molecular effects of drugs that prevent retinal capillary demise in the diabetic rat. RESEARCH DESIGN AND METHODS We examined the gene expression profile of retinal vessels isolated from rats with 6 months of streptozotocin-induced diabetes and compared it with that of control rats. We then tested whether the aldose reductase inhibitor sorbinil and aspirin, which have different mechanisms of action, prevented common molecular abnormalities induced by diabetes. The Affymetrix GeneChip Rat Genome 230 2.0 array was complemented by real-time RT-PCR, immunoblotting, and immunohistochemistry. RESULTS The retinal vessels of diabetic rats showed differential expression of 20 genes of the transforming growth factor (TGF)-β pathway, in addition to genes involved in oxidative stress, inflammation, vascular remodeling, and apoptosis. The complete loop of TGF-β signaling, including Smad2 phosphorylation, was enhanced in the retinal vessels, but not in the neural retina. Sorbinil normalized the expression of 71% of the genes related to oxidative stress and 62% of those related to inflammation. Aspirin had minimal or no effect on these two categories. The two drugs were instead concordant in reducing the upregulation of genes of the TGF-β pathway (55% for sorbinil and 40% for aspirin) and apoptosis (74 and 42%, respectively). CONCLUSIONS Oxidative and inflammatory stress is the distinct signature that the polyol pathway leaves on retinal vessels. TGF-β and apoptosis are, however, the ultimate targets to prevent the capillary demise in diabetic retinopathy.

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 (AR(-/-)) 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 AR(-/-) mice. Under short-term diabetic conditions, AR(-/-) 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 AR(-/-) mice were significantly lower than those in diabetic AR(+/+) mice. In addition, signs of oxidative stress, such as increased activation of c-Jun NH(2)-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 AR(-/-) mice, indicating that the diabetic AR(-/-) mice were protected from oxidative stress in the sciatic nerve. The diabetic AR(-/-) 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 AR(-/-) 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 AR(-/-) 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.

The objective of this study was to determine whether the polyol pathway enzyme aldose reductase mediates diabetes abnormalities in vascular smooth muscle cell (SMC) growth. Aldose reductase inhibitors (tolrestat or sorbinil) or antisense aldose reductase mRNA prevented hyperproliferation of cultured rat aortic SMCs induced by high glucose. Cell cycle progression in the presence of high glucose was blocked by tolrestat, which induced a G0-G1 phase growth arrest. In situ, diabetes increased SMC growth and intimal hyperplasia in balloon-injured carotid arteries of streptozotocin-treated rats, when examined 7 or 14 days after injury. Treatment with tolrestat (15 mg · kg−1 · day−1) diminished intimal hyperplasia and decreased SMC content of the lesion by 25%. Although tolrestat treatment increased immunoreactivity of the lesion with antibodies raised against protein adducts of the lipid peroxidation product 4-hydroxy trans-2-nonenal, no compensatory increase in lesion fibrosis was observed. Collectively, these results suggest that inhibition of aldose reductase prevents glucose-induced stimulation of SMC growth in culture and in situ. Even though inhibition of aldose reductase increases vascular oxidative stress, this approach may be useful in preventing abnormal SMC growth in vessels of diabetic patients.

Activation of protein kinase C (PKC) has been linked to the development of secondary diabetes complications. However, the underlying molecular mechanisms remain unclear. We examined the contribution of aldose reductase, which catalyzes the first, and the rate-limiting, step of the polyol pathway of glucose metabolism, to PKC activation in vascular smooth muscle cells (VSMCs) isolated from rat aorta and exposed to high glucose in culture. Exposure of VSMCs to high glucose (25 mmol/l), but not iso-osmotic mannitol, led to an increase in total membrane-associated PKC activity, which was prevented by the aldose reductase inhibitors tolrestat or sorbinil or by the ablation of aldose reductase by small interfering RNA (siRNA). The VSMCs were found to express low levels of sorbitol dehydrogenase, and treatment with the sorbitol dehydrogenase inhibitor CP-166572 did not prevent high-glucose-induced PKC activation. Stimulation with high glucose caused membrane translocation of conventional (alpha, beta1, beta2, and gamma) and novel (delta and epsilon) isoforms of PKC. Inhibition of aldose reductase prevented membrane translocation of PKC-beta2 and -delta and delayed the activation of PKC-beta1 and -epsilon, whereas membrane translocation of PKC-alpha and -gamma was not affected. Treatment with tolrestat prevented phosphorylation of PKC-beta2 and -delta. High glucose increased the formation of diacylglycerol (DAG) and enhanced phosphorylation of phospholipase C-gamma1 (PLC-gamma1). Inhibition of aldose reductase prevented high glucose-induced DAG formation and phosphorylation of PLC-gamma1 and PLC-beta2 and -delta. Inhibition of phospholipid hydrolysis by D609, but not by the synthetic alkyl-1-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycerophosphocholine, or edelfosine, prevented DAG formation. Treatment with sorbinil decreased the levels of reactive oxygen species in high-glucose-stimulated VSMCs. Hence, inhibition of aldose reductase, independent of sorbitol dehydrogenase, appears to be effective in diminishing oxidative stress and hyperglycemic changes in signaling events upstream to the activation of multiple PKC isoforms and PLC-gamma1 and may represent a useful approach for preventing the development of secondary vascular complications of diabetes.

This study evaluated the effects of aldose reductase inhibition on diabetes-induced oxidative-nitrosative stress and poly(ADP-ribose) polymerase (PARP) activation. In animal experiments, control and streptozotocin-induced diabetic rats were treated with or without the aldose reductase inhibitor (ARI) fidarestat (16 mg · kg−1 · day−1) for 6 weeks starting from induction of diabetes. Sorbitol pathway intermediate, but not glucose, accumulation in sciatic nerve and retina was completely prevented in diabetic rats treated with fidarestat. Sciatic motor nerve conduction velocity, hindlimb digital sensory nerve conduction velocity, and sciatic nerve concentrations of two major nonenzymatic antioxidants, glutathione and ascorbate, were reduced in diabetic versus control rats, and these changes were prevented in diabetic rats treated with fidarestat. Fidarestat prevented the diabetes-induced increase in nitrotyrosine (a marker of peroxynitrite-induced injury) and poly(ADP-ribose) immunoreactivities in sciatic nerve and retina. Fidarestat counteracted increased superoxide formation in aorta and epineurial vessels and in in vitro studies using hyperglycemia-exposed endothelial cells, and the DCF test/flow cytometry confirmed the endothelial origin of this phenomenon. Fidarestat did not cause direct inhibition of PARP activity in a cell-free system containing PARP and NAD+ but did counteract high-glucose–induced PARP activation in Schwann cells. In conclusion, aldose reductase inhibition counteracts diabetes-induced nitrosative stress and PARP activation in sciatic nerve and retina. These findings reveal the new beneficial properties of fidarestat, thus further justifying the ongoing clinical trials of this specific, potent, and low-toxic ARI.

We explored the specific impact of polyol pathway hyperactivity on dorsal root ganglia (DRG) using transgenic mice that overexpress human aldose reductase because DRG changes are crucial for the development of diabetic sensory neuropathy. Littermate mice served as controls. Half of the animals were made diabetic by streptozotocin injection and followed for 12 weeks. After diabetes onset, diabetic transgenic mice showed a significant elevation of pain sensation threshold after transient decrease and marked slowing of motor and sensory nerve conduction at the end of the study, while these changes were modest in diabetic littermate mice. Protein kinase C (PKC) activities were markedly reduced in diabetic transgenic mice, and the changes were associated with reduced expression of membrane PKC-α isoform that was translocated to cytosol. Membrane PKC-βII isoform expression was contrariwise increased. Calcitonin gene-related peptide–and substance P–positive neurons were reduced in diabetic transgenic mice and less severely so in diabetic littermate mice. Morphometric analysis disclosed neuronal atrophy only in diabetic transgenic mice. Treatment with an aldose reductase inhibitor (fidarestat 4 mg · kg−1 · day−1, orally) corrected all of the changes detected in diabetic transgenic mice. These findings underscore the pathogenic role of aldose reductase in diabetic sensory neuropathy through the altered cellular signaling and peptide expressions in DRG neurons.

This study examined the role of p38 mitogen-activated protein (MAP) kinase in transducing high glucose into deficits in nerve conduction velocity (NCV) that are characteristic of diabetic neuropathy. p38 activation and NCV were measured in streptozocin-induced diabetic rats treated with a p38 inhibitor, an aldose reductase inhibitor, and insulin. Dorsal root ganglia (DRG) from diabetic animals showed marked activation of p38 at 12 weeks of diabetes. Insulin treatment for the last 4 of 12 weeks of diabetes normalized p38 activation. Furthermore, activation was completely prevented by 12 weeks’ treatment with the aldose reductase inhibitor, fidarestat. Immunocytochemistry localized activation of p38 to the nuclei of virtually all sensory neuronal phenotypes in the DRG, and activation was clear in diabetes, as was inhibition by fidarestat and by the p38 inhibitor SB 239063. In the ventral horn of the spinal cord, p38 was present in motoneuron cell bodies; and again, activation in diabetes and fidarestat inhibition was clear. Treatment of diabetic animals with a specific inhibitor of p38 (SB 239063), fidarestat, or insulin also prevented reductions in both motor and sensory NCV. These findings suggest that increased polyol pathway flux in diabetic animals leads to the activation of p38. This activation can mediate changes in gene transcription and cellular phenotype that are likely to underlie the NCV deficits. Insulin and aldose reductase inhibitors can prevent excess polyol pathway flux, and hence these agents may prevent NCV deficits by preventing p38 MAP kinase activation.

With diabetes reaching epidemic proportions, there is an urgent need for reliable treatment strategies for microvascular (retinopathy, neuropathy, and nephropathy) and macrovascular (coronary heart disease and stroke) diabetes complications. The role of diabetic hyperglycemia in the development of complications has been extensively studied and a number of biochemical pathways activated by hyperglycemia have been identified. Among these, the polyol pathway is perhaps the most investigated and the most controversial pathway. The initial discovery by Ruth van Heyningen (1) of high sorbitol levels in diabetic rat lenses over 50 years ago followed by studies by Jin Kinoshita (2) laid the foundation for the polyol hypothesis in the pathogenesis of diabetes complications. This hypothesis asserts that diabetes complications result, in part, from direct or indirect consequences of sorbitol production from excess glucose by aldose reductase (AR). AR catalyzes the reduction of glucose to sorbitol, the accumulation of which has been postulated to alter the metabolism of myo -inositol, a six-carbon cyclic polyol, in nerve, ocular, and renal tissues (3,4). Depletion of myo -inositol reduces its incorporation into cellular phosphoinositide pools, resulting in chronic elevation of diacylglycerol (DAG), the chief physiological activator of protein kinase C (PKC) (5). Increased oxidation of sorbitol to fructose may also alter …

The peripheral nerve of experimental diabetic neuropathy (EDN) is reported to be ischemic and hypoxic, with an increased dependence on anaerobic metabolism, requiring increased energy substrate stores. When glucose stores become reduced, fiber degeneration has been reported. We evaluated glucose uptake, nerve energy metabolism, the polyol pathway, and protein kinase C (PKC) activity in EDN induced by streptozotocin. Control and diabetic rats received lipoic acid (0, 10, 25, 50, 100 mg/kg). Duration of diabetes was 1 month, and alpha-lipoic acid was administered intraperitoneally 5 times per week for the final week of the experiment. Nerve glucose uptake was reduced to 60, s 37, and 30% of control values in the sciatic nerve, L5 dorsal root ganglion, and superior cervical ganglion (SCG), respectively, in rats with EDN. Alpha-lipoic acid supplementation had no effect on glucose uptake in normal nerves at any dose, but reversed the deficit in EDN, with a threshold between 10 and 25 mg/kg. Endoneurial glucose, fructose, sorbitol, and myo-inositol were measured in sciatic nerve. Alpha-lipoic acid had no significant effect on either energy metabolism or polyol pathway of normal nerves. In EDN, endoneurial glucose, fructose, and sorbitol were significantly increased, while myo-inositol was significantly reduced. Alpha-lipoic acid had a biphasic effect: it dose-dependently increased fructose, glucose, and sorbitol, peaking at 25 mg/kg, and then fell beyond that dose, and it dose-dependently increased myo-inositol. Sciatic nerve cytosolic PKC was increased in EDN. ATP, creatine phosphate, and lactate were measured in sciatic nerve and SCG. Alpha-lipoic acid prevented the reduction in SCG creatine phosphate. We conclude that glucose uptake is reduced in EDN and that this deficit is dose-dependently reversed by alpha-lipoic acid, a change associated with an improvement in peripheral nerve function.

Diabetes increases the incidence of cardiovascular disease as well as the complications of myocardial infarction. Studies using animal models of diabetes have demonstrated that the metabolic alterations occurring at the myocyte level may contribute to the severity of ischemic injury in diabetic hearts. Of the several mechanisms being investigated to understand the pathogenesis of diabetic complications, the increased metabolism of glucose via the polyol pathway has received considerable attention. Deviant metabolic regulation due to increased flux through aldose reductase in diabetic hearts may influence the ability of the myocardium to withstand ischemia insult. To determine if aldose reductase inhibition improves tolerance to ischemia, hearts from acute type I diabetic and nondiabetic control rats were isolated and retrograde perfused. Each group was exposed to 1 micromol/l zopolrestat, a specific inhibitor of aldose reductase, for 10 min, followed by 20 min of global ischemia and 60 min of reperfusion in the absence of zopolrestat. Zopolrestat reduced sorbitol levels before ischemia in diabetic hearts. The cytosolic redox state (NADH/NAD+), as measured by lactate-to-pyruvate ratios, was significantly lowered under baseline, ischemic, and reperfusion conditions in diabetic hearts perfused with zopolrestat. In these diabetic hearts, ATP was significantly higher in zopolrestat hearts during ischemia, as were phosphocreatine and left ventricular-developed pressure on reperfusion. Zopolrestat provided similar metabolic and functional benefits in nondiabetic hearts. Creatine kinase release was reduced by approximately 50% in both nondiabetic and diabetic hearts treated with zopolrestat. These data indicate that inhibition of aldose reductase activity preserves high-energy phosphates, maintains a lower cytosolic NADH/NAD+ ratio, and markedly protects both diabetic and nondiabetic hearts during ischemia and reperfusion.

Oleanolic acid (OA), a natural component of many plant food and medicinal herbs, is endowed with a wide range of pharmacological properties whose therapeutic potential has only partly been exploited until now. Throughout complex and multifactorial mechanisms, OA exerts beneficial effects against diabetes and metabolic syndrome. It improves insulin response, preserves functionality and survival of β-cells, and protects against diabetes complications. OA may directly modulate enzymes connected to insulin biosynthesis, secretion, and signaling. However, its major contributions appear to be derived from the interaction with important transduction pathways, and many of its effects are consistently related to activation of the transcription factor Nrf2. Doing that, OA induces the expression of antioxidant enzymes and phase II response genes, blocks NF-κB, and represses the polyol pathway, AGEs production, and hyperlipidemia. The management of type 2 diabetes requires an integrated approach, which includes the early intervention to prevent or delay the disease progression, and the use of therapies to control glycemia and lipidemia in its late stages. In this sense, the use of functional foods or drugs containing OA is, undoubtedly, an interesting path.

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.

Enhanced metabolism of glucose via the polyol pathway may play an important role in the pathogenesis of diabetic retinopathy, neuropathy, and nephropathy. Aldose reductase catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step in the polyol pathway. Interruption of the polyol pathway by inhibition of aldose reductase holds considerable promise as a therapeutic measure to prevent or delay the onset and severity of these late complications of diabetes. Dramatic advances in our understanding of the molecular biology, enzymology, and three-dimensional structure of aldose reductase have occurred in recent years, providing new and challenging insights into the enzyme's catalytic mechanism. Recent developments in structure determination of aldose reductase and the implications for evaluation and development of aldose reductase inhibitors are summarized.

We investigated the effects of glucose on specific D-alpha-tocopherol binding to cultured bovine aortic endothelial cells. Our results confirmed that cultured bovine aortic endothelial cells have specific binding sites for D-alpha-tocopherol. These binding sites exhibited time- and temperature-dependent saturation. The specific binding affinity of D-alpha-tocopherol was significantly lower in endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 7 days compared with cells cultured in a physiological glucose concentration (5.6 mM). No significant reduction occurred in D-alpha-tocopherol binding when 11.1 mM mannitol was added to cells cultured in 5.6 mM glucose. The addition of an aldose reductase inhibitor (ICI-128436, Statil) did not significantly affect the high-glucose-induced reduction of D-alpha-tocopherol binding, although it reduced sorbitol levels in the cells compared with those from cells cultured in high concentrations of glucose. Moreover, significantly higher amounts of lipid peroxides were produced in aortic endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 3 days compared with cells cultured in a physiological concentration of glucose. These results indicate that high concentrations of glucose reduce D-alpha-tocopherol binding through mechanisms independent of putative osmotic effects of sorbitol accumulation in the cells. Possible mechanisms include glycation of protein or oxidative damage of cells and/or redox and metabolic imbalances associated with increased flux of glucose via the sorbitol pathway. A glucose-mediated reduction in D-alpha-tocopherol binding could diminish the beneficial effects of D-alpha-tocopherol to vascular endothelial cells and thereby may increase the vascular toxicity of hyperglycemia in diabetes mellitus.

Although the enhanced activity of the polyol pathway has been detected in diabetic glomeruli, the intraglomerular localization of this pathway has not yet been well defined. In this study, we attempted to identify aldose reductase, a key enzyme of the polyol pathway, in cultured rat mesangial cells and to characterize the properties of this enzyme using enzymological and immunological methods. When the aldose reductase (DL-glyceraldehyde-reducing) activity was analyzed in mesangial cell extract, the Lineweaver-Burk plot showed concave downward curvature, and the Michaelis constant was 0.83 mM DL-glyceraldehyde, and this activity was noncompetitively inhibited by an aldose reductase inhibitor, ICI-128,436. The enzyme activity was enhanced by the addition of sulfate ion and partially suppressed by barbital. The enzyme cross-reacted with the antisera against rat lens and testis aldose reductases on Ouchterlony plate, and migrated to the region of molecular weight of about 36,500 Da on Western blotting. The presence of aldose reductase mRNA was also confirmed by Northern analysis using cDNA for rat aldose reductase, 10Q. From these results, it was concluded that the aldose reductase may exist in rat glomerular mesangial cells and may play a role in the development of diabetic glomerulopathy, though the coexistence of aldehyde reductase(s) may not be fully ruled out.

Measurements were made of the levels of metabolic intermediates and activities of enzymes of the glycolytic route, pentose phosphate pathway, and polyol pathway in livers and kidneys of NOD mice. A 34% decrease in UDP-glucose, a 40% decrease in glucose-6-phosphate (G6P) and fructose-6-phosphate, and a 75% decrease in fructose-2,6-bisphosphate (F2,6P) were found in the livers of NOD mice. The fall in the level of F2,6P (the important regulator of glycolysis) is accompanied by a 20% reduction in the activity of phosphofructokinase. These changes are in agreement with previously reported liver depletion of glycogen and reduced synthesis of proteins and nucleic acids in the diabetic state. In the kidney, the increase in hexokinase activity is consistent with increased levels of G6P and glycogen content of kidney in diabetes. The decreased level of phosphoribosyl pyrophosphate was reported to be a regulator of kidney growth in the initial period of diabetes but can still be found in NOD mice 6 wk after development of hyperglycemia. The reported changes are similar to those seen in alloxan- or streptozocin-induced diabetic animals, but certain changes are more marked in NOD mice, especially those directed to increase nucleic acid and protein synthesis in the diabetic kidney.

Increased flux through the polyol pathway mediated by the enzyme aldose reductase may be associated with the development of diabetic neuropathy. Fifty-four diabetic patients (median age 56 yr, range 25-65 yr) with chronic neuropathic symptoms were randomly allocated to placebo or aldose reductase inhibition (300 or 600 mg ponalrestat ICI 128436) groups for 24 wk. Patients with vibration perception thresholds (VPTs) greater than 35 V at the great toe or thermal difference thresholds (TTs) greater than 10 degrees C on the dorsum of the foot were excluded from the trial. No significant changes were observed in symptoms of pain, numbness, or paresthesia between ponalrestat and placebo groups, and there were no improvements in VPT or TT at several sites. Posterior tibial nerve conduction velocity changed from 35.3 +/- 4.9 m/s at baseline to 33.4 +/- 4.0 m/s at 24 wk (NS) with placebo compared with 37.6 +/- 5.6 vs. 37.2 +/- 8.7 m/s (NS) with 300 mg ponalrestat and 34.5 +/- 6.1 vs. 36.2 +/- 6.8 m/s (NS) with 600 mg ponalrestat. Further studies are indicated with intervention at an earlier stage in the evolution of neuropathy and for longer periods.

The preventive effect of the aldose reductase inhibitor (ARI) ponalrestat on heart-rate variability and the development of autonomie neuropathy in the vagus nerve was investigated in the spontaneously diabetic BB rat. ARI treatment completely prevented the characteristic decrease in heart-rate variability and axonal atrophy of the vagus nerve for 4 mo in hyperglycemic BB rats. After 6 mo of treatment, the preventive effect on heart-rate variability was partial, and the vagus nerve demonstrated an increase in regenerating myelinated and unmyelinated fibers. These data suggest that autonomie neuropathy involving the vagus nerve is metabolically induced by demonstrating that inhibition of the polyol pathway significantly delays the occurrence of functional and structural autonomie neuropathy despite the presence of hyperglycemia.

Hyperalgesia to noxious stimuli is accompanied by increased spinal cyclooxygenase (COX)-2 protein in diabetic rats. The present studies were initiated to establish causality between increased spinal COX-2 activity and hyperalgesia during diabetes and to assess the potential involvement of polyol pathway activity in the pathogenesis of spinally mediated hyperalgesia. Rats with 1, 2, or 4 weeks of streptozotocin-induced diabetes exhibited significantly increased levels of spinal COX-2 protein and activity, along with exaggerated paw flinching in response to 0.5% paw formalin injection. Increased flinching of diabetic rats was attenuated by intrathecal pretreatment with a selective COX-2 inhibitor immediately before formalin injection, confirming the involvement of COX-2 activity in diabetic hyperalgesia. Chronic treatment with insulin or ICI222155, an aldose reductase inhibitor (ARI) previously shown to prevent spinal polyol accumulation and formalin-evoked hyperalgesia in diabetic rats, prevented elevated spinal COX-2 protein and activity in diabetic rats. In contrast, the ARI IDD676 had no effect on spinal polyol accumulation, elevated spinal COX-2, or hyperalgesia to paw formalin injection. In the spinal cord, aldose reductase immunoreactivity was present solely in oligodendrocytes, which also contained COX-2 immunoreactivity. Polyol pathway flux in spinal oligodendrocytes provides a pathogenic mechanism linking hyperglycemia to hyperalgesia in diabetic rats.

Previously studied inhibitors of aldose reductase were largely from two chemical classes, spirosuccinamide/hydantoins and carboxylic acids. Each class has its own drawbacks regarding selectivity, in vivo potency, and human safety; as a result, the pathogenic role of aldose reductase in diabetic retinopathy remains controversial. ARI-809 is a recently discovered aldose reductase inhibitor (ARI) of a new structural class, pyridazinones, and has high selectivity for aldose versus aldehyde reductase. To further test the possible pathogenic role of aldose reductase in the development of diabetic retinopathy, we examined the retinal effects of this structurally novel and highly selective ARI in insulinized streptozotocin-induced diabetic rats. ARI-809 treatment was initiated 1 month after diabetes induction and continued for 3 months at a dose that inhibited the polyol pathway in the retina of diabetic rats to a similar extent as sorbinil, a poorly selective hydantoin ARI previously shown to prevent retinopathy in this model. ARI-809 improved survival, inhibited cataract development, normalized retinal sorbitol and fructose, and protected the retina from abnormalities that also occur in human diabetes: neuronal apoptosis, glial reactivity, and complement deposition. Because ARI-809 is a novel chemotype highly selective for aldose reductase, these results support the notion that aldose reductase is the key relay that converts hyperglycemia into glucose toxicity in neural and glial cell types in the retina.

Activation of the polyol pathway has been linked to the development of secondary diabetic complications. However, the underlying molecular mechanisms remain unclear. To probe the contribution of this pathway, we examined whether inhibition of aldose reductase, which catalyzes the first step of the pathway, affects hyperglycemia-induced activation of the inflammatory transcription factor nuclear factor (NF)-kappaB. Treatment of vascular smooth muscle cells with the aldose reductase inhibitors tolrestat and sorbinil prevented high-glucose-induced protein kinase C (PKC) activation, nuclear translocation of NF-kappaB, phosphorylation of IKK, and the increase in the expression of intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and aldose reductase. High-glucose-induced NF-kappaB activation was also prevented by the PKC inhibitors chelerythrine and calphostin C. Ablation of aldose reductase by small interference RNA (siRNA) prevented high-glucose-induced NF-kappaB and AP-1 activation but did not affect the activity of SP-1 or OCT-1. Stimulation with iso-osmotic mannitol activated NF-kappaB and increased the expression of aldose reductase but not ICAM-1 and VCAM-1. Treatment with aldose reductase inhibitors or aldose reductase siRNA did not affect mannitol-induced NF-kappaB or AP-1 activation. Administration of tolrestat (15 mg . kg(-1) . day(-1)) decreased the abundance of activated NF-kappaB in balloon-injured carotid arteries of diabetic rats. Collectively, these results suggest that inhibition of aldose reductase, which prevents PKC-dependent nonosmotic NF-kappaB activation, may be a useful approach for treating vascular inflammation caused by diabetes.

The polyol (sorbitol) pathway of glucose metabolism is activated in many cell types when intracellular glucose concentrations are high, and it can generate cellular stress through several mechanisms. The role of the polyol pathway in the pathogenesis of diabetic retinopathy has remained uncertain, in part because it has been examined preferentially in galactose-induced retinopathy and in part because inhibition studies may not have achieved full blockade of the pathway. Having observed that the streptozotocin-induced diabetic rat accurately models many cellular processes characteristic of human diabetic retinopathy, we tested in the diabetic rat if documented inhibition of the polyol pathway prevents a sequence of retinal vascular abnormalities also present in human diabetes. An inhibitor of aldose reductase, the rate-limiting enzyme in the pathway, prevented the early activation of complement in the wall of retinal vessels and the decreased levels of complement inhibitors in diabetic rats, as well as the later apoptosis of vascular pericytes and endothelial cells and the development of acellular capillaries. Both rat and human retinal endothelial cells showed aldose reductase immunoreactivity, and human retinas exposed to high glucose in organ culture increased the production of sorbitol by a degree similar to that observed in the rat. Excess aldose reductase activity can be a mechanism for human diabetic retinopathy.

Diabetes is known to affect cataract formation by means of osmotic stress induced by activated aldose reductase in the sorbitol pathway. In addition, alterations in the bioavailability of numerous extralenticular growth factors has been reported and shown to result in various consequences. We have found that the basic fibroblast growth factor (bFGF) accumulates in the vitreous humor of 3- and 8-week diabetic rats. Consequently, the associating signal transduction cascades were severely disrupted, including upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and the common stress-associated mitogen-activated protein kinases p38 and SAPK/JNK. Conversely, under diabetic condition, we observed a dramatic inhibition of phosphatidylinositol-3 kinase activity in lenses obtained from the same animal. Rats treated with the aldose reductase inhibitor AL01576 for the duration of the diabetic condition showed that the diabetes-induced lenticular signaling alterations were normalized, comparable to controls. However, treatment of AL01576 in vitro was ineffective at normalizing the altered constituents in extracted diabetic vitreous after the onset of diabetes. The effect of AL01576 in the high galactose-induced cataract model in vitro was also examined. Administration of AL01576 to lens organ culture normalized the aberrant signaling effects and morphological characteristics associated with in vitro sugar cataract formation. In conclusion, our findings demonstrate diabetes-associated alterations in the lens signal transduction parameters and the effectiveness of AL01576 at normalizing such alterations. The causes for these alterations can be attributed to elevated vitreal bFGF in conjunction with osmotic stress and associated attenuation in redox status of the lens.

The study addressed the role for aldose reductase (AR) in 1) retinal oxidative stress and vascular endothelial growth factor (VEGF) overexpression in early diabetes, and 2) high glucose-induced oxidative stress in retinal endothelial cells. In vivo experiments were performed on control rats and diabetic rats treated with or without low or high dose of the AR inhibitor (ARI) fidarestat (2 or 16 mg · kg−1 · day−1). In vitro studies were performed on bovine retinal endothelial cells (BREC) cultured in either 5 or 30 mmol/l glucose with or without 1 μmol/l fidarestat. Intracellular reactive oxygen species were assessed using the 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe and flow cytometry. Both low and high doses of fidarestat (i.e., the doses that partially and completely inhibited sorbitol pathway hyperactivity) arrested diabetes-induced retinal lipid peroxidation. This was achieved due to upregulation of the key antioxidative defense enzyme activities rather than changes in reduced glutathione, oxidized glutathione, ascorbate and dehydroascorbate concentrations, and the glutathione and ascorbate redox states. Diabetes-associated 2.1-fold VEGF protein overexpression (enzyme-linked immunosorbent assay; ELISA) was dose-dependently prevented by fidarestat, whereas total VEGF mRNA and VEGF-164 mRNA (RT-PCR) abundance were not affected by either diabetes or the ARI. In BREC, fidarestat corrected hyperglycemia-induced increase in H2DCFDA fluorescence but not oxidative stress caused by three different pro-oxidants in normoglycemic conditions. In conclusion, increased AR activity contributes to retinal oxidative stress and VEGF protein overexpression in early diabetes. The findings justify the rationale for evaluation of fidarestat on diabetic retinopathy.

We tested the hypothesis that the apoptosis of inner retina neurons and increased expression of glial fibrillary acidic protein (GFAP) observed in the rat after a short duration of diabetes are mediated by polyol pathway activity. Rats with 10 weeks of streptozotocin-induced diabetes and GHb levels of 16 ± 2% (mean ± SD) showed increased retinal levels of sorbitol and fructose, attenuation of GFAP immunostaining in astrocytes, appearance of prominent GFAP expression in Müller glial cells, and a fourfold increase in the number of apoptotic neurons when compared with nondiabetic rats. The cells undergoing apoptosis were immunoreactive for aldose reductase. Sorbinil, an inhibitor of aldose reductase, prevented all abnormalities. Intensive insulin treatment also prevented most abnormalities, despite reducing GHb only to 12 ± 1%. Diabetic mice, known to have much lower aldose reductase activity in other tissues when compared with rats, did not accumulate sorbitol and fructose in the retina and were protected from neuronal apoptosis and GFAP changes in the presence of GHb levels of 14 ± 2%. This work documents discrete cellular consequences of polyol pathway activity in the retina, and it suggests that activation of the pathway and “retinal neuropathy” require severe hyperglycemia and/or high activity of aldose reductase. These findings have implications for how to evaluate the role of the polyol pathway in diabetic retinopathy.

Increased glucose utilization by aldose reductase (AR) has been implicated in the development of diabetes complications. However, the mechanisms that regulate AR during diabetes remain unknown. Herein we report that several nitric oxide (NO) donors prevent ex vivo synthesis of sorbitol in erythrocytes obtained from diabetic or nondiabetic rats. Compared with erythrocytes of nondiabetic rats, the AR activity in the erythrocytes of diabetic rats was less sensitive to inhibition by NO donors or by AR inhibitors—sorbinil or tolrestat. Treatment with NG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthesis, enhanced AR activity and sorbitol accumulation in tissues of nondiabetic rats. Application of transdermal nitroglycerin patches or treatment with l-arginine did not inhibit AR activity or sorbitol accumulation in the tissues of nondiabetic animals. Treatment with l-NAME increased, whereas treatment with l-arginine or nitroglycerine patches decreased AR activity and sorbitol content in tissues of diabetic rats. These observations suggest that NO maintains AR in an inactive state and that this repression is relieved in diabetic tissues. Thus, increasing NO availability may be a useful strategy for inhibiting the polyol pathway and preventing the development of diabetes complications.

Diabetes is a major risk factor for the development of atherosclerosis. In addition to increased risk of stroke, myocardial infarction, and peripheral vascular disease, diabetics suffer from a particularly aggressive form of atherosclerosis with greater in-hospital mortality following myocardial infarction and a higher incidence of heart failure, if they survive (1–3). While diabetics often have other accompanying risk factors for atherosclerosis (e.g., hypertension hypercholesterolemia, obesity), the additional risk conferred by diabetes and the particularly aggressive vascular and myocardial disease that affects diabetics suggest that diabetes-associated atherosclerosis involves unique pathogenic mechanisms. The systemic metabolic disturbances of diabetes, including hyperglycemia and hyperlipidemia, likely play a central role in the pathogenesis of diabetes-associated atherosclerosis through the generation of oxidative stress. Hyperglycemia causes increased flux through the polyol pathway, formation of advanced glycation end products, activation of protein kinase C isoforms, and increased hexosamine pathway flux, all of which may contribute to increased oxidative stress (4–6). Excessive free fatty acids delivered to nonadipose tissues can lead to reactive oxygen species (ROS) formation through cycles of oxidative phosphorylation, activation of NADPH oxidase, and alterations in mitochondrial structure that precipitate ROS production (7–9). In addition to evidence for activation of these pathways in cultured endothelial cells, human studies support the notion of increased systemic oxidative stress in diabetic subjects in …

Experimental diabetic peripheral neuropathy (DPN) is marked by impaired nerve conduction velocity (NCV), reduced nerve blood flow (NBF), and a variety of metabolic abnormalities in peripheral nerve that have been variously ascribed to hyperglycemia, abnormal fatty acid metabolism, ischemic hypoxia, and/or oxidative stress. Some investigators propose that NCV slowing in experimental DPN can be explained entirely on the basis of nerve energy depletion secondary to reduced NBF. This article reports highly selective effects of administration of the antioxidant DL-alpha-lipoic acid (LA) to streptozotocin-injected diabetic rats. LA improved digital sensory but not sciatic-tibial motor NCV, corrected endoneurial nutritive but not composite NBF, increased the mitochondrial oxidative state without correcting nerve energy depletion, and enhanced the accumulation of polyol pathway intermediates without worsening myo-inositol or taurine depletion. These studies implicate oxidative stress as an important pathophysiological factor in experimental DPN. They reveal complex interrelationships among nerve perfusion, energy metabolism, osmolyte content, conduction velocity, and oxidative stress that may reflect the heterogeneous and compartmentalized composition of peripheral nerve.

In this report, we made use of sorbitol dehydrogenase (SDH)-deficient mutant mice (C57BL/LiA) to test whether there is a close correlation between the level of polyol accumulation and the degree of reduction in motor nerve conduction velocity (MNCV) associated with diabetes. The C57BL/LiA mouse has SDH deficiency due to a G-to-A mutation at the +1 position of intron 8, thus producing only aberrant SDH transcripts. These C57BL/LiA mice should have higher levels of polyol accumulation in the peripheral nerve because of the inability to further metabolize sorbitol to fructose. Here, we confirm by Western blot analysis and high-performance liquid chromatography that these mice lack SDH in the sciatic nerve and other various tissues, whereas normal mice possess SDH. These C57BL/LiA mice do not display any obvious phenotype that includes peripheral neuropathy in the normal laboratory environment and breed normally as described previously, although the tissues that normally contain SDH accumulate more sorbitol. This finding suggested that C57BL/LiA mouse strain is a valid model for studying the role in diabetic neuropathy of the polyol pathway, which consists of two enzymes-aldose reductase for converting glucose to sorbitol and SDH for converting sorbitol to fructose. Sorbitol levels in the sciatic nerve of diabetic C57BL/10N, nondiabetic, and diabetic C57BL/LiA mice were increased 4.3-, 16.6-, and 38.1-fold, respectively, above that of nondiabetic C57BL/10N. The fructose level in the sciatic nerve was increased 2.4-fold in diabetic C57BL/10N mice compared with that of nondiabetic and diabetic C57BL/LiA mice. Diabetic SDH-deficient mice showed an MNCV reduction similar in magnitude to that of diabetic C57BL/10N mice, despite greater nerve sorbitol accumulation and the lack of fructose in the former. The present data suggest that the levels of sorbitol and fructose in the sciatic nerve of mice do not correlate with the severity of MNCV deficit associated with diabetes.

Reduced nerve perfusion is an important factor in the etiology of diabetic neuropathy. Studies in streptozotocin-induced diabetic rats show that nerve conduction velocity (NCV) and blood flow deficits are corrected by treatment with vasodilator drugs, with angiotensin II and endothelin-1 antagonists being particularly important. The AT1 antagonist ZD7155 also prevents diabetic deficits in regeneration following nerve damage, indicating that hypoperfusion is an important limitation for nerve repair. Metabolic changes include high polyol pathway flux, increased advanced glycosylation, elevated oxidative stress, and impaired ω-6 essential fatty acid metabolism. Aldose reductase inhibitors (ARIs) restore NCV via their effects on perfusion. ARI action probably depends on blocking the conversion of glucose to sorbitol, thus preventing depletion of vasa nervorum glutathione, an important endogenous free radical scavenger. Free radicals cause vascular endothelium damage and reduced nitric oxide vasodilation. Inhibition of advanced glycosylation and autoxidation (autoxidative glycosylation), major sources of free radicals, by aminoguanidine or transition metal chelators, corrects neurovascular dysfunction. Evening primrose oil supplies γ-linolenic acid (GLA) to improve vasodilator eicosanoid synthesis in diabetes, correcting nerve blood flow and NCV deficits. Interactions between some of these mechanisms have therapeutic implications. Thus, combined ARI and evening primrose oil treatment produced a 10-fold amplification of NCV and blood flow responses. Similarly, GLA effects are markedly enhanced when given in combination with ascorbate as ascorbyl-GLA. Thus, metabolic abnormalities combine to produce deleterious changes in nerve perfusion that make a major contribution to the etiology of diabetic neuropathy. The potential importance of multi-action therapy is stressed.

The impact of exaggerated polyol pathway flux on ciliary neurotrophic factor (CNTF)-like bioactivity and expression of CNTF in rat sciatic nerve was examined after 2 months of galactose intoxication. Polyol content was elevated (P < 0.001) and motor nerve conduction velocity reduced (P < 0.05) in galactose-fed rats compared with control animals or control and galactose-fed rats treated with the aldose reductase inhibitor (ARI) Ponalrestat. CNTF-like bioactivity in the galactose-fed group was reduced to 30% of that assayed in the control group (P < 0.001). ARI treatment significantly increased CNTF-like bioactivity by 60% compared with the untreated galactose group (P < 0.05) but did not restore it to control levels. Unexpectedly, bioactivity in ARI-treated control animals was increased by nearly 250% compared with untreated controls (P < 0.005). In addition to the deficit in CNTF bioactivity in untreated galactose rats, the expression of protein, but not of mRNA, was reduced (P < 0.05). In ARI-treated control and galactose-fed rats, the expression of CNTF peptide was significantly enhanced above control levels (both P < 0.05). Concomitant with the reduction in CNTF levels, there was a shift in the axonal size-frequency distribution of myelinated fibers toward smaller axons in galactose-fed rats that was prevented by ARI treatment. Since galactose feeding has little impact on levels of CNTF mRNA, these observations suggest that deficits in CNTF-like bioactivity may result from a posttranscriptional modification of neurotrophic protein expression or turnover. Unlike other functional and structural disorders in galactose neuropathy, factors other than polyol accumulation may contribute to the deficit in CNTF-like bioactivity.

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/1 glucose (HG) for 5–7 days was reduced by 48% vs. 5.5 mmol/1 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 μmol/1 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.

ext to the debate as to whether chronic hyperglycemia is the primary cause of the late vascular complications of diabetes, the longest running controversy among researchers and clinicians studying this disease is the role of the sorbitol pathway, particularly its initial enzymatic step, the reduction of glucose to sorbitol by aldose reductase in the pathogenesis of these complications. Because the outcome of the Diabetes Control and Complications Trial (DCCT) (1) appears to have effectively resolved the first issue, the aldose reductase controversy now takes center stage. The sorbitol pathway hypothesis is not the only mechanism for the pathogenesis of the complications of diabetes to have achieved serious consideration. Among others, for example, nonenzymatic glycation of proteins (2) is a plausible, if still largely untested, possibility. But over the more than 30 years since the British biochemist, Ruth vari Heyningen, first demonstrated an excess of sorbitol in the lenses of diabetic rats (3), a substantial accumulation of evidence has supported the sorbitol pathway as the principal actor in at least some of the late complications of diabetes in humans and experimental animals. 'This view is not unchallenged, however. In this discussion, I will summarize some of the evidence supporting the role of aldose reductase in producing several of the late complications of diabetes, but also point out those contradictory findings that render the sorbitol pathway hypothesis controversial.

Acetyl-L-carnitine reduces the latencies of electroretinogram oscillatory potentials in healthy humans. The effect of acetyl-L-carnitine (50 mg · kg−1 · day−1) on the increased electroretinogram latencies found in rats with STZ-induced hyperglycemia of 3-wk duration was evaluated. The aldose reductase inhibitor sorbinil, which has been shown to normalize abnormal electroretinogram tracings associated with STZ-induced diabetes, was used as a positive control. Aldose reductase inhibitors are thought to lower tissue sorbitol while increasing myo-inositol. The electroretinograms of the STZ-induced diabetic rats in this study were abnormal; treatment with acetyl-L-carnitine as well as sorbinil significantly improved electroretinogram b-wave amplitude and decreased the latencies of oscillatory potentials 2 and 3. Acetyl-L-carnitine treatment of STZ-induced diabetic rats did not affect hyperglycemia or erythrocyte polyol pathway activity as reflected by erythrocyte sorbitol levels. In contrast, sorbinil did reduce elevated erythrocyte sorbitol levels. This suggests that the impaired electroretinograms associated with STZ-induced diabetes may not be caused solely by increased polyol pathway activity.

In diabetic subjects, polyol pathway activity might inhibit neutrophil function and cause nerve damage. The effects of ponalrestat, an aldose reductase inhibitor, were assessed on neutrophil intracellular killing of Escherichia coli and on autonomic function in diabetic subjects in a randomized double-blind, placebo-controlled, crossover trial. We studied 31 diabetic subjects with autonomic dysfunction and 21 age- and sex-matched control subjects. During two 12-wk treatment periods, the diabetic subjects took either 600 mg of ponalrestat or matching placebo once daily. Neutrophil killing of E. coli was measured by a microbiological assay technique. Kmax by neutrophils from the diabetic subjects was lower than in the control group (Kmax of diabetic subjects 54.5 ± 26.4 vs. control subjects 67.3 ± 16.3, P = 0.045). Ponalrestat significantly increased bacterial killing in the diabetic subjects (Kmax of ponalrestat 75.1 ± 16.5 vs. placebo 58.2 ± 20.8, P = 0.003) so that there was no longer any significant difference in Kmax between the control subjects and the diabetic subjects on active treatment. Ponalrestat had no significant effect on a range of standard cardiovascular autonomic nerve function tests. We conclude that neutrophil killing of E. coli is impaired in diabetic subjects with autonomic dysfunction. This is restored to normal by ponalrestat.

The polyol pathway is present in tissues of several organs where its activation may participate in the development of diabetic complications. We measured the accumulation of polyol-pathway intermediates in HPT cells isolated from 21 different human kidneys from nondiabetic individuals. When exposed to 27.5 mM glucose in the growth media, cells isolated from ∼75% of individuals (accumulators) accumulated sorbitol within 1–4 days, whereas 25% (nonaccumulators) accumulated only negligible amounts, even when the period of exposure was extended to 2 wk. Surprisingly, measurement of the activities of the polyol-pathway enzymes showed no difference in the levels of either AR or SDH between accumulators and nonaccumulators, even when the conversion of galactose to galactitol was used to measure AR activity in intact cells independently of SDH. Measurement of sorbitol in the growth media indicated that nonaccumulators were not releasing sorbitol into the growth media. Fructose levels in the conditioned growth media were 4 times higher in the sorbitol-accumulating cells. Together, these results indicate that the tendency of cells from an individual to accumulate significant amounts of sorbitol may reflect the cells' ability to metabolize sorbitol in steps subsequent to the polyol pathway.

Hyperglycemia causes enhanced glucose metabolism by the polyol pathway in tissues not requiring insulin for glucose uptake. It has been suggested that the high level of aldose reductase activity may cause functional and structural abnormalities in diabetes and may be involved in the development of late complications. To elucidate the effect of an aldose reductase inhibitor (ponalrestat) on kidney function in uncomplicated insulin-dependent diabetes mellitus (IDDM), 20 normoalbuminuric IDDM patients were randomized to follow either 6 mo of treatment with ponalrestat ( n = 11, mean ± SD age 30 ± 8 yr, diabetes duration 10 ± 6 yr) or 6 mo of placebo (age 33 ± 7 yr, diabetes duration 12 ± 6 yr). The glomerular filtration rate (clearance of [ 125 I]iothalamate) was significantly reduced from 140 ± 18 to 129 ± 10 ml · min −1 · 1.73 m −2 , 2 P = 0.02) in the ponalrestat-treated patients, whereas no change was seen after placebo (142 ± 12 vs. 141 ± 12 ml · min −1 · 1.73 m −1 ). The renal plasma flow (clearance of 131 I-labeled hippuran), urinary albumin excretion rate (radioimmunoassay), fractional albumin clearance, and renal vascular resistance were unchanged in both groups. HbA 1c showed a modest increase during ponalrestat (7.9 ± 1.8 vs. 8.7 ± 1.5%, 2 P = 0.01) but was unchanged during placebo. No side effects of ponalrestat were observed. Thus, inhibition of aldose reductase may reduce the characteristic hyperfiltration in uncomplicated IDDM.

Early functional disturbances in nerve, retina, and lens in diabetes mellitus appear to result from a common mechanism involving increased polyol-pathway activity with an associated effect on tissue myo-inositol metabolism. We tested the role of increased polyol-pathway activity in the early glomerular hemodynamic abnormalities in experimental diabetes in rats with dietary myo-inositol supplementation or the administration of sorbinil, an aldose reductase inhibitor. Each maneuver prevented the glomerular hyperfiltration of early streptozocin-induced diabetes and reversed the hyperfiltration of established diabetes of 10 days' duration. We also found that the abnormal response to captopril in diabetic rats was improved by dietary myo-inositol supplementation or sorbinil administration. Although nonhypotensive doses of captopril lowered glomerular filtration rate (GFR) in diabetic rats on a 0.01% myo-inositol diet, GFR increased substantially after captopril infusion in diabetic rats treated with sorbinil or myo-inositol supplementation. These data suggest that normalization of tissue myo-inositol metabolism restores normal responsiveness to angiotensin II; this may contribute to the reduction in GFR with the two experimental maneuvers. We also tested the interaction between polyol-pathway activation and high dietary protein intake. Aldose reductase inhibition and dietary myo-inositol supplementation had no effect on the component of increased GFR due to 50% dietary protein intake but specifically inhibited the hyperfiltration attributable to diabetes. These results suggest that hyperglycemia acts through increased polyol-pathway activity and its effects on tissue myo-inositol metabolism to play a fundamental role in the pathogenesis of the glomerular hyperfiltration characteristic of early diabetes.

To test the general applicability of the hypothesis that diabetes mellitus causes increased polyol pathway activity, decreased tissue free myo-inositol, and resultant pathological changes in tissues susceptible to the ravages of diabetes, we measured glucose, sorbitol, and myo-inositol with quantitative histochemical techniques in layers of the cornea, the aortic myointima, the cardiac left ventricle and atrioventricular node (AVN), and retina and kidney after 19 days or 2 mo (mildly diabetic non-insulin-treated [MD] and severely diabetic insulin-treated [SD] groups) in the alloxan-induced diabetes model. In the aqueous humor, glucose rose linearly with increased serum glucose, sorbitol was markedly increased in the MD and SD groups, and myo-inositol did not change in any diabetic group. There was no change in glucose or sorbitol in aortic myointima in any group, but myo-inositol was decreased in 19-day diabetic rabbits by 26%, unchanged in MD rabbits but paradoxically increased by 60% in SD rabbits. Glucose, sorbitol, and myo-inositol increased in all three corneal layers in SD rabbits but only in epithelium and stroma in 19-day and MD rabbits. AVN glucose and sorbitol did not change in 19-day diabetic, MD, or SD diabetic rabbits. AVN myo-inositol was three times higher than ventricular myo-inositol and did not appear to change in SD rabbits. Retinal pigmented epithelium myo-inositol was decreased 30% in SD rabbits. Glomerular myo-inositol was also decreased, but not significantly, in SD rabbits. We conclude that the paradoxical increase in corneal and aortal myo-inositol raises fundamental questions about the general applicability of the myo-inositol-depletion hypothesis.

The human proximal tubule (HPT) is the characteristic site within the kidney that mediates absorption of glucose. This study was designed to determine whether cultured HPT cells would respond to a hyperglycemic environment through activation of the polyol pathway. The results of this study clearly indicate that exposure of the HPT cells to an extracellular glucose concentration greater than or equal to 11 mM results in substantial intracellular accumulation of sorbitol. This accumulation is inhibited by approximately 70% by treatment with 100 microM sorbinil. When cells growing 24 h on 27.5 mM glucose were changed to medium containing 5.5 mM glucose, sorbitol concentration returned to the control level within 12 h. The activity of aldose reductase was increased by a factor of 1.6 by exposure to elevated glucose concentrations, and the relative reactivity of the enzyme with glucose as substrate was approximately 0.1 compared with that of glyceraldehyde as substrate. Together, these results indicate that cultured cells derived from the HPT undergo activation of the polyol pathway when exposed to a hyperglycemic environment.

We measured motor nerve conduction velocity (MNCV), Na(+)-K(+)-ATPase activity, polyol-pathway metabolites, and myo-inositol in sciatic nerves from control mice, galactose-fed (20% wt/wt diet) mice, and galactose-fed mice given the aldose reductase inhibitor ponalrestat (300-mg/kg diet). Treatments were maintained for 4 wk. Galactose feeding was associated with a 21.5% reduction in MNCV (P less than 0.001), which was almost completely prevented by ponalrestat. Galactose-fed mice showed an 81% increase in Na(+)-K(+)-ATPase (P less than 0.01), an effect completely prevented by aldose reductase inhibition. Treatment of a separate galactose-fed group with sorbinil (300 mg/kg diet) also attenuated the MNCV deficit and prevented the increased Na(+)-K(+)-ATPase activity associated with galactosemia. Accumulation of galactitol in the nerves of galactose-fed mice was prevented by aldose reductase inhibition, but there were no alterations in myo-inositol levels in the sciatic nerves of any group. These data show that exaggerated flux through the polyol pathway can cause an MNCV deficit that is unrelated to either myo-inositol levels or NA(+)-K(+)-ATPase activity.

A method to measure the polyol pathway metabolic flux in the intact rabbit lens by 13C nuclear magnetic resonance spectroscopy is described. In the lens exposed to 35.5 mM glucose, the polyol pathway accounts for ⅓ of the total glucose turnover. The high metabolic activity of the pathway suggests a significant alteration in the reduced to oxidized pyridine nucleotide ratio in the lens exposed to high glucose.

To assess whether the ultrastructure and composite metabolism of the arterial wall are subject to acute modification by exposure to an elevated glucose concentration, paired samples of the intact rabbit aortic intima-media preparation were incubated with 5 mM or 20 mM glucose for 1 h under conditions in which the ultrastructural and metabolic stability of samples incubated with 5 mM glucose has previously been demonstrated. Incubation with 20 mM glucose did not alter the qualitative electron-microscopic appearance of the tissue, and no significant changes were found on a quantitative morphometric evaluation of the aortic endothelium. A detectable, composite-free intracellular glucose concentration was not demonstrable in samples incubated with 5 mM or 20 mM glucose by a comparison of the ratio of glucose space:inulin space. Samples incubated with 5 mM glucose had significant sorbitol concentrations and unexpectedly high fructose concentrations but did not release fructose into the medium at detectable rates. Incubation with 20 mM glucose resulted in an increase in tissue sorbitol without an increase in tissue fructose or a detectable rate of fructose release. Tissue water content and myoinositol concentration were unaltered by incubation with 20 mM glucose, as were the rates of O2 uptake and lactate production whether the gas phase was 5% CO2:95% O2 or 5% CO2:95% air. The ultrastructure' of intact aortic intima-media is not acutely altered by exposure to an elevated glucose concentration. Ambient glucose concentration is not the primary determinant of composite-free intracellular glucose concentration in arterial wall, and an increase in medium glucose concentration does not result in a significant increase in glucose utilization via the polyol pathway or induce the concomitants of increased polyol pathway activity demonstrable in a conventionally prepared aortic intima-media preparation.

Decreased glutathione levels in the ocular lens have been invoked as a possible cause for the decreased lenticular Na+-K+-ATPase in diabetes because both are corrected by aldose reductase inhibitors, and the Na+-K+-ATPase is known to be susceptible to oxidation inactivation. Because an analogous Na+-K+-ATPase defect that is prevented by aldose reductase inhibitors has been described in diabetic peripheral nerve, we examined the effect of streptozocin (STZ) diabetes and aldose reductase inhibition on reduced (GSH) and oxidized (GSSG) glutathione levels in crude homogenates of rat sciatic nerve. Neither GSSG nor GSH levels were altered by 2 or 8 wk of untreated diabetes or by aldose reductase inhibition. Because the defect in Na+-K+-ATPase is fully expressed by 4 wk of STZ diabetes, we conclude that altered glutathione redox state plays no detectable role in the pathogenesis of this defect in diabetic peripheral nerve.

The rapid conversion of glucose to sorbitol by aldose reductase and the consequent hyperosmolarity of the cytoplasm has been shown to be the primary cause of the so-called “sugar” or “osmotic” cataract in many animal lenses. It is not as clear, however, that hyperosmolarity is the principal factor in the etiology of cataracts in human diabetic subjects. In fact, the comparatively low activity of aldose reductase in the human lens as compared with several animal lenses, and the osmotically insignificant levels of sorbitol pathway products (sorbitol and fructose), suggest that hyperosmolarity, per se, may not be as important a factor in human cataract formation as it is in animals. We present evidence that the flux of glucose and sorbitol through the rat lens is markedly reduced by oxidative stress (0.1 mM H2O2). Sorbitol accumulation is reduced by 114%, sorbitol turnover is reduced by 78%, sorbitol production is reduced by 90%, fructose accumulation is reduced by 60%, and fructose turnover is reduced by 76% in the presence of 36 mM glucose. H2O2 does not affect glucose turnover, the glucose rate constant, or the ATP level significantly at 36 mM glucose, but at 5.5 mM glucose, 0.2 mM H2O2 leads to a rapid loss of ATP that can be prevented by 0.04 mM sorbinil, an aldose reductase inhibitor. These results suggest that inhibition of aldose reductase by sorbinil renders rat lenses better able to cope with oxidative stress. In the absence of an aldose reductase inhibitor, elevating ambient glucose may render a lens less able to scavenge oxidants by diverting NADPH into sorbitol production. The importance of the rate of flux of glucose through the sorbitol pathway, rather than the absolute concentration of sorbitol or fructose, is stressed in considering the mechanisms underlying complications of diabetes mellitus.

Although enhanced activity of the polyol pathway has been implicated in the pathogenesis of certain complications of diabetes, evidence that aldose reductase activity and sorbitol content are increased in the characteristic tissue site of the diabetic renal lesion has been lacking. We therefore measured polyols in glomeruli isolated from control and streptozotocin-diabetic rats, and assessed whether changes in diabetic glomeruli could be prevented by oral administration of the aldose reductase inhibitor sorbinil. Compared with control, polyol content of glomeruli isolated from diabetic rats was increased 10-fold and fourfold at 6 and 9 wk, respectively, after induction of diabetes, but was unchanged in glomeruli from rats treated with sorbinil throughout the experimental periods. In contrast, glomerular myo-inositol content was reduced in diabetic samples; this fall in myo-inositol levels was also completely prevented by sorbinil. These results establish that glomeruli contain aldose reductase activity and provide the first demonstration that glomerular polyol content increases while myo-inositol content decreases in diabetes and that oral sorbinil prevents these changes despite persistent hyperglycemia.