d-α-Tocopherol prevents the hyperglycemia induced activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular smooth muscle cell by an increase of DAG kinase activity

Abstract

We have reported that d-a-tocopherol can prevent hyperglycemia-induced activation of DAG and PKC levels in vascular tissues as well as normalizing retinal blood flow and renal hyperfiltration. The mechanism of this effect, however, is not clear. Aside from a-tocopherol’s principal role as an antioxidant agent, it has also been shown to act as a membrane stabilizer. Another possibility is that the effect of a-tocopherol is focused on the activation of DAG kinase, which is a key enzyme in the metabolism of DAG. Therefore, in this study, we examined the effect of a-tocopherol on the DAG kinase activity in vascular smooth muscle cell. We have also examined the effect of a-tocopherol, its analogues, and probucol on DAG kinase activities and expression. The present study showed that d-a-tocopherol’s inhibitory effect on DAG-PKC pathway is by increasing DAG kinase activity in rat and human vascular smooth muscle cell (VSMC). Total DAG level was increased by 40910% (mean9S.E.) (PB0.05) in human VSMC, after exposure to 22 vs 5 mM glucose. This increase was normalized by d-a-tocopherol treatment in a concentration-dependent manner. In parallel, DAG kinase activation by d-a-tocopherol was also induced in a timeand dose-dependent manner. DAG kinase activity was increased by 57919% (PB0.05) in human VSMC and 112935% (PB0.05) in rat VSMC after 24 h of incubation with d-a-tocopherol (100 mg:ml). Another lipophilic antioxidant, probucol, also increased DAG kinase activity by 124934%, but other vitamin E analogues with much less antioxidant potencies were ineffective. Western blots of various DAG kinase isoforms were not changed by d-a-tocopherol treatment. These results provide strong and detailed evidence that d-a-tocopherol can prevent hyperglycemia induced DAG-PKC activation by enhancing DAG kinase activity, probably through an antioxidant effect. © 1999 Elsevier Science Ireland Ltd. All rights reserved.