Thomas C. Hohman

Comparison of the effects of inhibitors of aldose reductase and sorbitol dehydrogenase on neurovascular function, nerve conduction and tissue polyol pathway metabolites in streptozotocin-diabetic rats

Summary Aldose reductase inhibitors (ARIs) attenuate diabetic complications in several tissues, including lens, retina, kidney, blood vessels, striated muscle and peripheral nerve. However, it is unclear whether their action in diabetes mellitus depends directly on inhibiting the conversion of glucose to sorbitol by aldose reductase or indirectly by reducing the sorbitol available for subsequent metabolism to fructose by sorbitol dehydrogenase. To identify the polyol pathway step most relevant to complications, particularly neuropathy, we compared the biochemical effects of a sorbitol dehydrogenase inhibitor, WAY-135 706, (250 mg · kg−1· day−1) and an ARI, WAY-121 509, (10 mg · kg−1· day−1) on a variety of tissues, and their effects on nerve perfusion and conduction velocity. After 6 weeks of untreated streptozotocin diabetes, rats were treated for 2 weeks. Sorbitol was elevated 2.1–32.6-fold by diabetes in lens, retina, kidney, aorta, diaphragm, erythrocytes and sciatic nerve; this was further increased (1.6–8.2-fold) by WAY-135 706 whereas WAY-121 509 caused a marked reduction. Fructose 1.6–8.0-fold elevated by diabetes in tissues other than diaphragm, was reduced by WAY-135 706 and WAY-121 509, except in the kidney. Motor and sensory nerve conduction velocities were decreased by 20.2 and 13.9 %, respectively with diabetes. These deficits were corrected by WAY-121 509, but WAY-135 706 was completely ineffective. A 48.6 % diabetes-induced deficit in sciatic nutritive endoneurial blood flow was corrected by WAY-121 509, but was unaltered by WAY-135 706. Thus, despite profound sorbitol dehydrogenase inhibition, WAY-135 706 had no beneficial effect on nerve function. The data demonstrate that aldose reductase activity, the first step in the polyol pathway, makes a markedly greater contribution to the aetiology of diabetic neurovascular and neurological dysfunction than does the second step involving sorbitol dehydrogenase. [Diabetologia (1997) 40: 271–281]

Protein kinase C effects on nerve function, perfusion, Na+,K+-ATPase activity and glutathione content in diabetic rats

Aims//hypothesis. Increased protein kinase C activity has been linked to diabetic vascular complications in the retina and kidney, which were attenuated by protein kinase C antagonist treatment. Neuropathy has a vascular component, therefore, the aim was to assess whether treatment with WAY151 003 or chelerythrine, inhibitors of protein kinase C regulatory and catalytic domains respectively, could correct nerve blood flow, conduction velocity, Na+,K+-ATPase, and glutathione deficits in diabetic rats. Methods. Diabetes was induced by streptozotocin. Sciatic nerve conduction velocity was measured in vivo and sciatic endoneurial perfusion was monitored by microelectrode polarography and hydrogen clearance. Glutathione content and Na+,K+-ATPase activity were measured in extracts from homogenised sciatic nerves. Results. After 8 weeks of diabetes, sciatic blood flow was 50 % reduced. Two weeks of WAY151 003 (3 or 100 mg/kg) treatment completely corrected this deficit and chelerythrine dose-dependently improved nerve perfusion. The inhibitors dose-dependently corrected a 20 % diabetic motor conduction deficit, however, at high doses ( > 3.0 mg/kg WAY151003; > 0.1 mg/kg chelerythrine) conduction velocity was reduced towards the diabetic level. Sciatic Na+,K+-ATPase activity, 42 % reduced by diabetes, was partially corrected by low but not high dose WAY151 003. In contrast, only a very high dose of chelerythrine partially restored Na+,K+-ATPase activity. A 30 % diabetic deficit in sciatic glutathione content was unchanged by protein kinase C inhibition. The benefits of WAY151 003 on blood flow and conduction velocity were blocked by nitric oxide synthase inhibitor co-treatment. Conclusion/interpretation. Protein kinase C contributes to experimental diabetic neuropathy by a neurovascular mechanism rather than through Na+,K+-ATPase defects. [Diabetologia (1999) 42: 1120–1130]

Interactions between essential fatty acid, prostanoid, polyol pathway and nitric oxide mechanisms in the neurovascular deficit of diabetic rats.

SummaryImpaired Ω-6 essential fatty acid metabolism and exaggerated polyol pathway flux contribute to the neurovascular abnormalities in streptozotocin-diabetic rats. The potential interactions between these mechanisms were examined by comparing the effects of threshold doses of aldose reductase inhibitors and evening primrose oil, alone and in combination, on neurovascular deficits. In addition, highdose aldose reductase inhibitor and evening primrose oil treatment effects were challenged by co-treatment with the cyclo-oxygenase inhibitor, flurbiprofen, or the nitric oxide synthase inhibitor, NG-nitro-l-arginine. Eight weeks of diabetes caused an 18.9% reduction in sciatic motor conduction velocity (p<0.001). This was only modestly ameliorated by a 0.1% dietary supplement of evening primrose oil or the aldose reductase inhibitors ZD5522 (0.25 mg · kg−1 · day−1) and WAY121509 (0.2 mg · kg−1· day−1) for the final 2 weeks. However, joint treatment with primrose oil and ZD5522 or WAY121509 caused marked 71.5 and 82.4% corrections, respectively, of the conduction deficit. Sciatic nutritive blood flow was 43.1% reduced by diabetes (p<0.001) and this was corrected by 67.8% with joint ZD5522 and primrose oil treatment (p<0.001). High-dose WAY121509 (10 mg · kg−1 · day−1) and primrose oil (10% dietary supplement) prevented sciatic conduction velocity and nutritive blood flow deficits in 1-month diabetic rats (p<0.001). However, these effects were abolished by flurbiprofen (5 mg · kg−1 · day−1) and NG-nitro-l-arginine (10 mg · kg−1 · day−1) co-treatment (p<0.001). Thus, the data provide evidence for synergistic interactions between polyol pathway/nitric oxide and essential fatty acid/cyclo-oxygenase systems in the control of neurovascular function in diabetic rats, from which a potential therapeutic advantage could be derived.

Effect of hyperglycemia and the aldose reductase inhibitor tolrestat on sural nerve biochemistry and morphometry in advanced diabetic peripheral polyneuropathy

Tolrestat is a well tolerated nonhydantoin aldose reductase inhibitor that has been reported to improve nerve conduction in diabetic animals and humans. Its effects on nerve biochemistry and structure have not been studied in patients with diabetic neuropathy. Patients with advanced diabetic neuropathy treated with long-term open-label tolrestat were randomly assigned to continuation on drug treatment or to placebo-controlled drug withdrawal for 12 months. At the end of this period, sural nerve biopsies were obtained for measurement of glucose, sorbitol, and fructose content, and for detailed morphometric analysis. Tolrestat ameliorated the glucose-mediated increase in sorbitol and fructose in sural nerve tissue. No statistically significant differences in nerve morphometry emerged between the two groups; however, both treatment groups exhibited increased nerve-fiber regeneration and normalization of axo-glial dysfunction and segmental demyelination following long-term tolrestat treatment. These findings are similar to those previously reported in a placebo-controlled sequential nerve biopsy study with the aldose reductase inhibitor sorbinil. Thus tolrestat is a biochemically effective aldose reductase inhibitor in human diabetic nerve with potential therapeutic efficacy for diabetic neuropathy.

Induction of aldose reductase expression in rat kidney mesangial cells and Chinese hamster ovary cells under hypertonic conditions.

Rat kidney cortex mesangial cells (MES) and Chinese hamster ovary cells (CHO) responded to hypertonicity (600 mosmol/kg) in culture by accumulating sorbitol. The accumulation of sorbitol was due to increased aldose reductase (AR) activity, apparently brought about by increased levels of AR mRNA and protein. The levels of AR mRNA increased approximately 60-fold in MES cells and 30-fold in CHO cells by 24 h in culture media (300 mosmol/kg supplemented with 150 mM NaCl, 600 mosmol/kg total). AR activity also markedly increased (14- to 16-fold above control), but MES took 4 days and CHO 6 days to reach this maximum. Other osmolytes, raffinose and sorbitol (at concentrations of 250 to 300 mM) elicited the same response as that of 150 mM NaCl. These data show that AR expression is induced in MES and CHO cells under hypertonic conditions. Of special interest is the induction of large amounts of AR in rat kidney cortex mesangial cells, a target tissue of diabetes and a site where excessive accumulation of sorbitol is suspected to be a critical factor in diabetic nephropathy.

Characterization of mRNA and genes for aldose reductase in rat

Aldose reductase (AR; E.C. 1. 1. 1. 21) has been implicated in a variety of diabetic complications. To investigate the expression of this enzyme in target tissues susceptible to such complications, mRNA encoding AR was characterized by Northern blot hybridization in various tissues and cultured cell preparations. The size of mRNA for AR (approximately 1500 bases) was in good agreement with the size determined by sequence analysis. A cDNA probe for AR from rat lens hybridized to the same size species of RNA isolated from cultured dog lens epithelial cells, cultured human retinal capillary pericytes (mural cells), and Y 79 human retinoblastoma cells. In rat tissues, a substantial amount of mRNA was expressed not only in lens, but also in retina, sciatic nerve and kidney medulla. AR mRNA seemed to be less abundant in rat skeletal muscle and brain, and was scarcely present in liver. Furthermore, Southern blot analysis of rat genomic DNA indicated that there are multiple sequences related to that for AR, probably indicating the existence of a multi-gene family.

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.

ATP-sensitive K(+) channel effects on nerve function, Na(+), K(+) ATPase, and glutathione in diabetic rats.

Abstract Some vasodilators correct nerve conduction velocity and endoneurial blood flow deficits in diabetic rats. It is not known whether vasa nervorum has ATP-sensitive K + (K ATP ) channels that mediate vasodilation, or whether K ATP channels could modulate peripheral nerve function. Therefore, we examined the effects of 2 weeks treatment with the K ATP channel openers, celikalim and WAY135201 (R-4-[3, 4-dioxo-2-(1, 2, 2-trimethyl-propylamino)-cyclobut-1-1-enylamino]-3-methoxy-benzonitrile), on sciatic nerve blood flow, conduction velocity, Na + –K + ATPase activity and glutathione content after 6 weeks of untreated streptozotocin-diabetes in rats. Blood flow and motor conduction velocity, 47.6% and 20.3% reduced by diabetes, respectively, were completely restored by both celikalim and WAY135201 treatments. Diabetes diminished sciatic Na + –K + ATPase activity by 47.6% and this was 80–90% corrected by the K ATP channel openers. Sciatic nerve glutathione content, 30.3% reduced by diabetes, was unaffected by celikalim or WAY135201. Thus, K ATP channel openers had marked beneficial effects on nerve perfusion and function in experimental diabetic neuropathy, and may be suitable for further study in clinical trials.

Reversal of defective peripheral nerve conduction velocity, nutritive endoneurial blood flow, and oxygenation by a novel aldose reductase inhibitor, WAY-121,509, in streptozotocin-induced diabetic rats

The main aim was to investigate whether 1 month of aldose reductase inhibitor treatment could correct a deficit in sciatic nerve nutritive blood flow following 1 month of untreated streptozotocin-induced diabetes in rats. Treatment was with two doses of WAY-121,509, both of which completely blocked neuronal sorbitol accumulation. The high dose fully corrected a motor conduction velocity deficit, whereas the low dose caused 51.3% amelioration. Nutritive endoneurial blood flow, monitored by hydrogen clearance, was 43.4% reduced after 1 month of diabetes. This was completely corrected by the high dose of WAY-121,509. In addition, vascular conductance was supranormal and there was a decrease in arteriovenous shunt flow. Low dose treatment caused a 55.6% improvement of the nutritive endoneurial blood flow deficit, paralleling the conduction velocity effect. WAY-121,509 did not alter nerve perfusion in nondiabetic rats. Data from multiple sciatic nerve penetrations by oxygen sensitive microelectrodes revealed a 42.0% deficit in mean endoneurial oxygen tension with diabetes, whereas tensions were in the nondiabetic range for high dose WAY-121,509 treatment. Thus, the data highlight neurovascular actions of aldose reductase inhibition, and suggest that neuronal polyol pathway metabolite levels are a poor predictor of functional efficacy.

Influence of tolrestat on the defective leukocyte–endothelial interaction in experimental diabetes

One of the most devastating secondary complications of diabetes is the blunted inflammatory response that becomes evident even in the very early stages of poorly controlled diabetes mellitus. While the etiology of this diminished response is not clearly understood, it has been linked to a decrease in the respiratory burst of neutrophils, as well as a decrease in microvessel response to inflammatory mediators and defective leukocyte-endothelial interactions. Using video microscopy to visualize vessels of the internal spermatic fascia, we have characterized leukocyte-endothelial interactions in alloxan-induced diabetic and in galactosemic rats by quantitating the number of leukocytes rolling along the venular endothelium and the number of leukocytes sticking to the vascular wall after topical application of zymosan-activated plasma or leukotriene B(4) (1 ng/ml), as well as after the application of a local irritant stimulus (carrageenan, 100 microg). We observed that while 33 days of alloxan-induced diabetes or 7 days of galactosemia had no effect on total or differential leukocyte counts and on the wall shear rate, both treatments significantly (P<0.001) reduced the number of leukocytes rolling along the venular endothelium by about 70% and the number of adhered leukocytes in postcapillary venules by 60%. These effects were not observed in diabetic and galactosemic animals treated with an aldose reductase inhibitor. The results suggest that impaired leukocyte-endothelial cell interactions are a consequence of an enhanced flux through the polyol pathway.