Reversal of redox-dependent inhibition of diacylglycerol kinase by antioxidants in mesangial cells exposed to high glucose.

Activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is one of the pathomechanisms of diabetic nephropathy. We previously reported that d-α-tocopherol, well known as an antioxidant, enhances diacylglycerol kinase (DGK) activity, leading to the reduction of excess DAG accumulation and PKC activation in the glomeruli of streptozotocin-induced diabetic rats. However, it remains to be determined whether the effect of d-α-tocopherol on DGK activity is exerted through its antioxidative action. DAG contents, PKC and membranous DGK activity were measured in cultured human glomerular mesangial cells under normal (5.5 mM) or high glucose (27.5 mM) conditions in the presence or absence of two antioxidants (50 μM d-α-tocopherol and probucol). Mesangial cells were exposed to hydrogen peroxide (H2O2) (10-1000 μM) in the presence or absence of 300 U/ml catalase, followed by measurement of DGK activity. Both antioxidants restored the high glucose-induced decrease in DGK activity, resulting in the reduction of high glucose-induced activation of the DAG-PKC pathway. In mesangial cells exposed to H2O2 at various concentrations, DGK activity decreased in a dose-dependent manner. The addition of antioxidative enzyme catalase to the cells reversed the H2O2-mediated down-regulation of DGK activity. In conclusion, DGK activity is reduced by oxidative stress in human mesangial cells cultured under high glucose conditions. Antioxidants, including d-α-tocopherol and probucol may improve hyperglycemia-induced DAG-PKC activation by enhancing DGK activity.