CP-166572

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 (α, β1, β2, and γ) and novel (δ and ε) isoforms of PKC. Inhibition of aldose reductase prevented membrane translocation of PKC-β2 and -δ and delayed the activation of PKC-β1 and -ε, whereas membrane translocation of PKC-α and -γ was not affected. Treatment with tolrestat prevented phosphorylation of PKC-β2 and -δ. High glucose increased the formation of diacylglycerol (DAG) and enhanced phosphorylation of phospholipase C-γ1 (PLC-γ1). Inhibition of aldose reductase prevented high glucose-induced DAG formation and phosphorylation of PLC-γ1 and PLC-β2 and -δ. 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-γ1 and may represent a useful approach for preventing the development of secondary vascular complications of diabetes.