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alpha-Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of alpha-tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with alpha-tocopherol for 24 h, the intracellular GSH was increased at every concentration of alpha-tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with alpha-tocopherol at 50 microM for 24 h exhibited resistance against H2O2. However, a short exposure of HaCaT keratinocytes to alpha-tocopherol for 1 h did not influence either the GSH level or the resistance to H2O2. These findings suggest that GSH, which is inductively synthesized by alpha-tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of alpha-tocopherol on the GSH level, BSO, which is a typical inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), was used. When BSO was added to HaCaT keratinocytes, no action of alpha-tocopherol on the GSH level was observed. On the other hand, alpha-tocopherol resulted in the up-regulation of gamma-GCS-HS (heavy subunit) mRNA. In addition, water soluble alpha-tocopherol derivatives (alpha-tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that alpha-tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of gamma-GCS-HS mRNA.

We previously reported that epidermal glycation causes an increase in saturated fatty acid (FA) content in a differentiated reconstructed skin model and HaCaT cells. However, the relationship between ceramides (CERs) and glycation and their effects on stratum corneum (SC) barrier function was not elucidated. In this study, we investigated the effect of glycation on lipid content in 6-day-old cultured reconstructed skin. We used the EPISKIN RHE 6D model and induced glycation using glyoxal. In addition to transepidermal water loss, content of CERs, cholesterol and FA in the reconstructed epidermal model were analyzed by high performance thin layer chromatography. Expression of genes related to ceramide metabolism was determined by real time RT-PCR. Membrane fluidity of stratum corneum lipid liposomes (SCLL) that mimic glycated epidermis was analyzed using an electron spin resonance technique. It was found that FA was significantly increased by glycation. CER[NS], [AP], and cholesterol were decreased in glycated epidermis. Expression of ceramide synthase 3 (CERS3) was significantly decreased while fatty acid elongase 3 was increased by glyoxal in a dose dependent manner. Membrane fluidity of SCLL mimicking the lipid composition of glycated epidermis was increased compared with controls. Therefore, disruption of CER and FA content in glycated epidermis may be regulated via CERS3 expression and contribute to abnormal membrane fluidity.