Increased protein kinase C (PKC) activity has been implicated in the pathogenesis of diabetic retinopathy and nephropathy. However, the role of PKC in diabetic neuropathy remains unclear. The present study was conducted to compare the effect of PKC inhibition by a PKC-beta-selective inhibitor, LY333531 (LY), on diabetic nerve dysfunction with that of an aldose reductase inhibitor, NZ-314 (NZ). Streptozotocin-induced diabetic rats were treated with or without LY and/or NZ for 4 weeks, and motor nerve conduction velocity (MNCV), coefficient of variation of R-R interval (CVR-R), sciatic nerve blood flow (SNBF), peak latencies of oscillatory potentials on electroretinogram, PKC activities in membranous and cytosolic fractions of sciatic nerves, and polyol contents in the tail nerves were measured. Untreated diabetic rats demonstrated delayed MNCV, decreased CVR-R, reduced SNBF, and prolonged peak latencies of oscillatory potentials. Treatment with LY as well as NZ prevented all these deficits in diabetic rats. There were no significant differences in PKC activities in membranous or cytosolic fractions of sciatic nerves between normal and diabetic rats. Treatment with neither LY nor NZ altered PKC activities. Nerve myo-inositol depletion in diabetic rats was ameliorated not only by NZ, but also by LY. These observations suggest that inhibition of PKC-beta by LY may have a beneficial effect in preventing the development of diabetic nerve dysfunction, and that this effect may be mediated through its action on the endoneurial micro-vasculature.

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Diabetes mellitus is one of the major risk factors for atherosclerosis and is associated with an increased incidence of coronary heart diseases and cerebrovascular diseases [1,2]. The high prevalence of these macrovascular diseases in diabetic patients can be explained by hyperglycaemia in itself [3] as well as by the increased frequency of conventional risk factors such as hypertension, hyperlipidemia, obesity, and smoking.The proliferation of vascular smooth muscle cells is one of the characteristic features of atherosclerosis [4]. According to previous reports [4,5], platelet-derived growth factor (PDGF) plays an important part in the accelerated proliferation of smooth muscle Diabetologia (2001) Abstract Aims/hypothesis. The protein kinase C (PKC), platelet-derived growth factor (PDGF) and polyol pathway play important parts in the hyperproliferation of smooth muscle cells, a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between polyol pathway, protein kinase C and platelet-derived growth factor in the development of diabetic macroangiopathy. Methods. Smooth muscle cells were cultured with 5.5 or 20 mmol/l glucose with or without an aldose reductase inhibitor, epalrestat, or a PKC-b specific inhibitor, LY333 531. Protein kinase C activities, the expression of PKC-bII isoform and PDGF-b receptor protein, free cytosolic NAD + :NADH ratio, the contents of reduced glutathione, and proliferation activities were measured. Results. Smooth muscle cells cultured with 20 mmol/l glucose showed statistically significant increases in protein kinase C activities, the expression of PKCbII isoform and PDGF-b receptor protein, and proliferation activities, compared with smooth muscle cells cultured with 5.5 mmol/l glucose. Although epalrestat and LY333 531 inhibited protein kinase C activation induced by glucose to the same degree, the effects of epalrestat on proliferation activities and expression of the PDGF-b receptor were more prominent than those of LY333 531. Epalrestat improved the glucose-induced decrease in free cytosolic NAD + :NADH ratio and reduced glutathione content, but LY333 531 did not. The increased expression of membranous PKC-bII isoform was normalized by epalrestat. Conclusion/interpretation. These observations suggest that polyol pathway hyperactivity contributes to the development of diabetic macroangiopathy through protein kinase C, PDGF-b receptor, and oxidative stress, and that an aldose reductase inhibitor has a therapeutic value for this complication. [Diabetologia (2001) 44: 480±487]

RESULTS -In diabetic patients not treated with epalrestat, the erythrocyte CML level was significantly elevated above levels seen in nondiabetic individuals (49.9 ± 5.0 vs. 31.0 ± 5.2 U/g protein, P Ͻ 0.05) and was significantly lower in patients receiving epalrestat (33.1 ± 3.8 U/g protein, P Ͻ 0.05). Similar results were observed with 3-DG. The treatment of patients with epalrestat for 2 months significantly lowered the level of erythrocyte CML (46.2 ± 5.6 at baseline vs. 34.4 ± 5.0 U/g protein, P Ͻ 0.01) along with erythrocyte 3-DG (P Ͻ 0.05), triosephosphates (P Ͻ 0.05), fructose (P Ͻ 0.05), sorbitol (P Ͻ 0.05), and plasma TBARS (P Ͻ 0.05) without changes in plasma glucose and HbA 1c levels. A positive correlation was evident between the erythrocyte CML and sorbitol (r = 0.49, P Ͻ 0.01) or fructose (r = 0.40, P Ͻ 0.05) levels in diabetic patients.CONCLUSIONS -The results indicate that epalrestat administration lowers CML and associated variables and that polyol metabolites are correlated with CML in the erythrocytes of diabetic patients. The observed results suggest that aldose reductase activity may play a substantial role in the intracellular formation of CML in the mediation of reactive intermediate metabolites and oxidative stress.